Humanized anti-ACTH antibodies and use thereof

ABSTRACT

The present invention is directed to antibodies and fragments thereof having binding specificity for ACTH. Embodiments of this invention relate to the binding fragments of antibodies described herein, comprising the sequences of the VH, VL and/or CDR polypeptides described herein, and the polynucleotides encoding them. The invention also contemplates anti-ACTH antibodies and binding fragments thereof conjugated to one or more functional or detectable moieties. The invention further contemplates methods of making said anti-ACTH antibodies and binding fragments thereof. Embodiments of the invention also pertain to the use of anti-ACTH antibodies and binding fragments thereof for the diagnosis, assessment, prevention and treatment of diseases and disorders associated with ACTH, such as CAH, FGD, Cushing&#39;s Disease, Cushing&#39;s Syndrome, Parkinson&#39;s disease, obesity, diabetes, sleep disorders, depression, anxiety disorders, cancer, muscle atrophy, hypertension, hyperinsulinemia, cognitive dysfunction, Alzheimer&#39;s disease, galactorrhea, stress related conditions, cardiac conditions, metabolic syndrome, hyperaldosteronism, Conn&#39;s syndrome and familial hyperaldosteronism.

RELATED APPLICATION DISCLOSURE

This application is a divisional of U.S. patent application Ser. No.16/101,761, filed Aug. 13, 2018, which is a divisional of U.S. patentapplication Ser. No. 14/974,908, filed Dec. 18, 2015, now U.S. Pat. No.10,047,157, which claims the benefit of U.S. Provisional Pat. Appl. Nos.62/094,805, filed Dec. 19, 2014, U.S. Provisional Pat. Appl. No.62/118,563, filed Feb. 20, 2015, U.S. Provisional Pat. Appl. No.62/207,284, filed Aug. 19, 2015, each and all of which is herebyincorporated by reference in its entirety.

SEQUENCE DISCLOSURE

The instant application contains a Sequence Listing, which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Dec. 9, 2019, isnamed “1143257o005605.txt” and is 681,264 bytes in size.

FIELD

This invention pertains to novel antibodies and antibody fragments,preferably chimeric, humanized or human antibodies and fragments thereofthat specifically bind to human adrenocorticotrophic hormone(hereinafter “ACTH”) and compositions containing these anti-ACTHantibodies and anti-ACTH antibody fragments. Preferably, such anti-ACTHantibodies or antibody fragments (i) will not substantially interactwith (bind) a polypeptide consisting of the 13 N-terminal amino acidresidues of ACTH (ACTH₁₋₁₃) and/or alpha-MSH, and/or (ii) the 22C-terminal amino acid residues of ACTH (ACTH₁₈₋₃₉) (Corticotropin-LikeIntermediate Peptide or CLIP). In addition, the invention relates tonucleic acids encoding said anti-ACTH antibodies and anti-ACTH antibodyfragments. Further, the invention pertains to the use of said nucleicacids to express said antibodies and antibody fragments in desired hostcells. Also, the invention pertains to anti-idiotypic antibodiesproduced against any of such antibodies.

The invention further relates to therapeutic and diagnostic uses ofanti-ACTH antibodies and antibody fragments, preferably chimeric,humanized or human antibodies and antibody fragments that specificallybind to ACTH that antagonize one or more ACTH-related activities in thetreatment or prophylaxis of diseases wherein the suppression ofACTH-related activities and/or the reduction of steroid, e.g., cortisol,corticosterone and/or aldosterone, levels are therapeutically orprophylactically desirable, including congenital adrenal hyperplasia(CAH), Classical CAH, Nonclassical CAH, familial glucocorticoiddeficiency (FGD), Cushing's disease, Cushing's Syndrome,hyperaldosteronism including primary hyperaldosteronism (such as Conn'ssyndrome) secondary hyperaldosteronism, and familial hyperaldosteronism,sleep apnea, obesity, diabetes, anxiety disorders, cognitivedysfunction, Alzheimer's disease, and other conditions disclosed herein.Preferably such antibodies or antibody fragments will not substantiallyinteract with (bind) a polypeptide consisting of the 13 N-terminal aminoacid residues of ACTH (ACTH₁₋₁₃) and/or alpha-MSH, or (ii) the 22C-terminal amino acid residues of ACTH (ACTH₁₈₋₃₉) (CLIP).

BACKGROUND

Adrenocorticotropic hormone (ACTH), a 39 amino acid peptide, is producedby cleavage of a large precursor molecule, pro-opiomelanocortin (POMC).Post-translational enzymatic processing of POMC yields otherbiologically active peptides (e.g., corticotropin-like intermediatepeptide (CLIP), melanocyte-stimulating hormone (MSH), and lipotrophin(LPH)) in addition to ACTH as a result of tissue-specific processing ofPOMC. See Bicknell, J. Neuroendocrinology 20: 692-99 (2008).

The POMC gene has been remarkably conserved throughout evolution. Avariety of organisms have a single functional copy of the gene with thesame overall gene structure. The POMC gene is predominantly expressed inthe anterior and intermediate lobes of the pituitary, and it isgenerally accepted that the majority of POMC peptides found in thecirculation are derived from the pituitary, whereas POMC peptidesproduced in extra-pituitary tissues (e.g., brain, lymphocytes, skin,testis, thyroid, pancreas, gut, kidney adrenal and liver) act in anautocrine or paracrine fashion. See Bicknell, J. Neuroendocrinology 20:692-99 (2008).

POMC peptides, including ACTH, are believed to act primarily throughmelanocortin receptors (MCRs), a family of five G protein-coupledreceptors (i.e., MC1R, MC2R, MC3R, MC4R and MC5R). MCRs are expressed indiverse tissues, and serve discrete physiological functions. MC1R, whichis expressed on melanocytes, macrophages and adipocytes, is involved inpigmentation and inflammation. MC2R, which is expressed in the adrenalcortex, is involved in adrenal steroidogenesis. MC3R, which is expressedin the central nervous system (CNS), gastrointestinal (GI) tract andkidney, is involved in energy homeostasis and inflammation. MC4R, whichis expressed in the CNS and spinal cord, is involved in energyhomeostasis, appetite regulation and erectile function. MC5R, which isexpressed on lymphocytes and exocrine cells, is involved in exocrinefunction and regulation of sebaceous glands. See Ramachandrappa et al.,Frontiers in Endocrinology 4:19 (2013).

MC2R is reported to be unique among the MCR family for being highlyspecific for ACTH. See, Mountjoy K G et al., Science 1992;257:1248-1251; and Schioth H B et al, Life Sci 1996; 59: 797-801.However, while MC3R is the only MCR with significant affinity forgamma-MSH, it can also bind alpha-MSH and ACTH with approximately equalaffinity. See Gantz I, et al., J Biol Chem 1993; 268: 8246-8250. Also,at extremely high plasma concentrations, ACTH can bind to and activateMC1R resulting in hyperpigmentation, e.g., observed in subjects withfamilial glucocorticoid deficiency (FGD) (Turan et al., “An atypicalcase of familial glucocorticoid deficiency without pigmentation causedby coexistent homozygous mutations in MC2R (T152K) and MC1R (R160W).” J.Clin. Endocrinol. Metab. 97E771-E774 (2012)).

ACTH, one of the major end-products of POMC processing, is a hormonethat is essential for normal steroidogenesis and the maintenance ofnormal adrenal weight. ACTH is secreted by the pituitary gland inresponse to physiological or psychological stress and its principaleffects are increased production and release of corticosteroids. Inparticular, ACTH is secreted from corticotropes in the anterior lobe (oradenohypophysis) of the pituitary gland in response to the release ofthe hormone corticotropin-releasing hormone (CRH) by the hypothalamus.Once secreted, ACTH then travels to the adrenal cortex, where it bindsto and activates MC2R. Activation of MC2R results in the production ofcAMP in the adrenal cell. cAMP binds and activates protein kinase (PKA),which activates the conversion of the lipid cholesterol to the steroidhormone cortisol.

Cortisol is a hormone that affects numerous biological processes inorder to restore homeostasis after stress. Exemplary processes regulatedby cortisol include regulating glucose homeostasis, increasing bloodpressure, gluconeogenesis, promoting metabolism of glycogen, lipids, andproteins, and suppressing the immune system. Under normal physiologicalconditions, cortisol levels are tightly regulated. However, in someconditions (including diseases and disorders further described herein),cortisol levels are elevated. The overproduction of cortisol has beenshown to have many negative effects, such as damaging the hippocampus, aregion of the brain that is critical for cognitive functions andregulation of the hypothalamus/pituitary/adrenal axis; increasing fatdeposits, blood pressure levels, and blood sugar levels; bone loss;muscle weakness; and suppression of the immune system. Therefore,elevated cortisol levels may play a role in ACTH-driven hypercortisolism(such as Cushing's Disease or Cushing's Syndrome), obesity, diabetes,sleep apnea, depression, anxiety disorders, cancer (such as Cushing'sSyndrome resulting from ectopic ACTH expression, e.g., in small celllung cancer, non-small cell lung cancer (NSCLC), pancreatic carcinoma,neural tumors, or thymoma), muscle atrophies, hypertension, cognitivedysfunction, galactorrhea and metabolic syndromes.

Aldosterone is a hormone released by the adrenal glands that helpsregulate blood pressure. In particular, aldosterone increases thereabsorption of sodium and water and the release of potassium in thekidneys. In some disease conditions, aldosterone levels are elevated.For example, primary and secondary hyperaldosteronism occur when theadrenal gland releases too much of the hormone aldosterone. Primaryhyperaldosteronism such as Conn's syndrome results from a problem withthe adrenal gland itself that causes the release of too muchaldosterone, whereas the excess aldosterone in secondaryhyperaldosteronism is caused by something outside the adrenal gland thatmimics the primary condition, e.g., by causing the adrenal gland torelease too much aldosterone. Primary hyperaldosteronism used to beconsidered a rare condition, but some experts believe that it may be thecause of high blood pressure in some patients. Most cases of primaryhyperaldosteronism are caused by a noncancerous (benign) tumor of theadrenal gland. The condition is most common in people ages 30-50 years.Secondary hyperaldosteronism is frequently due to high blood pressureand it may also be related to disorders such as cirrhosis of the liver,heart failure, and nephrotic syndrome. Therefore, elevated aldosteronelevels may play a role in hyperaldosteronism including primaryhyperaldosteronism (such as Conn's syndrome), secondaryhyperaldosteronism and familial hyperaldosteronism.

SUMMARY

The invention in general relates to human, humanized or chimerizedanti-human adrenocorticotrophic hormone (“ACTH”) antibodies or antibodyfragments. In one embodiment, the human, humanized or chimerizedanti-ACTH antibody or antibody fragment does not substantially interactwith (i.e., bind to) a polypeptide consisting of: (i) the 13 N-terminalamino acid residues of ACTH (ACTH₁₋₁₃) and/or alpha-MSH, and/or (ii) the22 C-terminal amino acid residues of ACTH (ACTH₁₈₋₃₉).

The human, humanized or chimerized anti-ACTH antibody or antibodyfragment may be selected from the group consisting of scFvs,camelbodies, nanobodies, IgNAR, Fab fragments, Fab′ fragments, MetMablike antibodies, monovalent antibody fragments, and F(ab′)₂ fragments.Additionally, the human, humanized or chimerized anti-ACTH antibody orantibody fragment may substantially or entirely lack N-glycosylationand/or O-glycosylation. In one embodiment, the human, humanized orchimerized anti-ACTH antibody or antibody fragment comprises a humanconstant domain, e.g., an IgG1, IgG2, IgG3, or IgG4 antibody. In anotherembodiment, the human, humanized or chimerized anti-ACTH antibody orantibody fragment comprises an Fc region that has been modified to alterat least one of effector function, half-life, proteolysis, orglycosylation. For example, the Fc region may contain one or moremutations that alters or eliminates N- and/or O-glycosylation.

In one embodiment, the human, humanized or chimerized anti-ACTH antibodyor antibody fragment binds to ACTH with a K_(D) of less than or equal to5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M,5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M,10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M. Preferably, the human, humanized orchimerized anti-ACTH antibody or antibody fragment binds to ACTH with aK_(D) of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, or 10⁻¹² M. More preferably, the human, humanized orchimerized anti-ACTH antibody or antibody fragment binds to ACTH with aK_(D) that is less than about 100 nM, less than about 10 nM, less thanabout 1 nM, less than about 100 pM, less than about 50 pM, less thanabout 40 pM, less than about 25 pM, less than about 1 pM, between about10 pM and about 100 pM, between about 1 pM and about 100 pM, or betweenabout 1 pM and about 10 pM. In exemplary embodiments the K_(D) value maybe detected by surface plasmon resonance (e.g., BIAcore®) at 25 or 37°C. However, other methods such as ELISA and KINEXA may alternatively beused.

In another embodiment, the human, humanized or chimerized anti-ACTHantibody or antibody fragment binds to ACTH with an off-rate (k_(d)) ofless than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻⁵ s⁻¹, or 10⁻⁵ s⁻¹.

In yet another embodiment, the human, humanized or chimerized anti-humanACTH antibody or antibody fragment that specifically binds to the linearor conformational epitope(s) and/or competes for binding to the samelinear or conformational epitope(s) on human ACTH as an anti-human ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H, preferably Ab2.H or Ab13.H. Inparticular, the human, humanized or chimerized anti-human ACTH antibodyor antibody fragment specifically binds to the same linear orconformational epitope(s) on human ACTH as an anti-human ACTH antibodyselected from the group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H,Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H,Ab13.H, Ab15.H, and Ab17.H, preferably Ab2.H or Ab13.H. The epitope(s)may be identified using a binding assay that detects the binding of saidanti-human ACTH antibody or antibody fragment to one or more peptides ina library of overlapping linear peptide fragments that span the fulllength of human ACTH. Preferably, the epitope is identified usingalanine scanning mutation strategy.

In some embodiments, the human, humanized or chimerized anti-human ACTHantibody or antibody fragment contains at least 2 complementaritydetermining regions (CDRs), at least 3 CDRs, at least 4 CDRs, at least 5CDRs or all six CDRs of an anti-human ACTH antibody selected from thegroup consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H. In exemplary embodiments, the antibody or fragment will retainthe V_(H) CDR3 and/or the V_(L) CDR3 of one of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, or Ab17.H.

In a specific embodiment, the human, humanized or chimerized anti-humanACTH antibody or antibody fragment comprises (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO:4; a CDR2 sequenceconsisting of SEQ ID NO:6; and a CDR3 sequence consisting of SEQ IDNO:8; and/or (b) a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:24; a CDR2 sequence consisting of SEQ ID NO:26;and a CDR3 sequence consisting of SEQ ID NO:28. Alternatively, theanti-human ACTH antibody or antibody fragment may comprise (a) avariable heavy chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:2 and/or(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:22. Preferably, the anti-human ACTH antibody or antibody fragmentcomprises (a) a variable heavy chain having the amino acid sequence ofSEQ ID NO:2, and/or (b) a variable light chain having the amino acidsequence of SEQ ID NO:22. More specifically, the anti-human ACTHantibody or antibody fragment may comprise (a) a heavy chain having theamino acid sequence of SEQ ID NO: 1, and/or (b) a light chain having theamino acid sequence of SEQ ID NO:21.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:44; aCDR2 sequence consisting of SEQ ID NO:46; and a CDR3 sequence consistingof SEQ ID NO:48; and/or (b) a variable light chain comprising a CDR1sequence consisting of SEQ ID NO:64; a CDR2 sequence consisting of SEQID NO:66; and a CDR3 sequence consisting of SEQ ID NO:68. Alternatively,the anti-human ACTH antibody or antibody fragment may comprise (a) avariable heavy chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:42, and/or(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:62. Preferably, the anti-human ACTH antibody or antibody fragmentcomprises (a) a variable heavy chain having the amino acid sequence ofSEQ ID NO:42, and/or (b) a variable light chain having the amino acidsequence of SEQ ID NO:62. More specifically, the anti-human ACTHantibody or antibody fragment may comprise (a) a heavy chain having theamino acid sequence of SEQ ID NO:41, and/or (b) a light chain having theamino acid sequence of SEQ ID NO:61.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:84; aCDR2 sequence consisting of SEQ ID NO:86; and a CDR3 sequence consistingof SEQ ID NO:88; and/or (b) a variable light chain comprising a CDR1sequence consisting of SEQ ID NO: 104; a CDR2 sequence consisting of SEQID NO: 106; and a CDR3 sequence consisting of SEQ ID NO: 108.Alternatively, the anti-human ACTH antibody or antibody fragment maycomprise (a) a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:82, and/or (b) a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO: 102. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:82, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO: 102. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:81, and/or (b) a lightchain having the amino acid sequence of SEQ ID NO:101.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO: 124; aCDR2 sequence consisting of SEQ ID NO: 126; and a CDR3 sequenceconsisting of SEQ ID NO: 128; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 144; a CDR2 sequenceconsisting of SEQ ID NO: 146; and a CDR3 sequence consisting of SEQ IDNO: 148. Alternatively, the anti-human ACTH antibody or antibodyfragment may comprise (a) a variable heavy chain comprising an aminoacid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO: 122 and/or (b) a variable light chain comprisingan amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO: 142. Preferably, the anti-human ACTHantibody or antibody fragment comprises (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 122, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO: 142. Morespecifically, the anti-human ACTH antibody or antibody fragmentcomprises (a) a heavy chain having the amino acid sequence of SEQ ID NO:121, and/or (b) a light chain having the amino acid sequence of SEQ IDNO: 141.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO: 164; aCDR2 sequence consisting of SEQ ID NO: 166; and a CDR3 sequenceconsisting of SEQ ID NO: 168; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 184; a CDR2 sequenceconsisting of SEQ ID NO: 186; and a CDR3 sequence consisting of SEQ IDNO: 188. Alternatively, the anti-human ACTH antibody or antibodyfragment may comprises (a) a variable heavy chain comprising an aminoacid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:162, and/or (b) a variable light chain comprisingan amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO:182. Preferably, the anti-human ACTHantibody or antibody fragment comprises (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO: 162, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO:182. Morespecifically, the anti-human ACTH antibody or antibody fragmentcomprises (a) a heavy chain having the amino acid sequence of SEQ IDNO:161, and/or (b) a light chain having the amino acid sequence of SEQID NO:181.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:204; aCDR2 sequence consisting of SEQ ID NO:206; and a CDR3 sequenceconsisting of SEQ ID NO:208; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:224; a CDR2 sequenceconsisting of SEQ ID NO:226; and a CDR3 sequence consisting of SEQ IDNO:228. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise (a) a variable heavy chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:202 and/or (b) a variable light chain comprisingan amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO:222. Preferably, the anti-human ACTHantibody or antibody fragment comprises (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO:202, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO:222. Morespecifically, the anti-human ACTH antibody or antibody fragmentcomprises (a) a heavy chain having the amino acid sequence of SEQ IDNO:201, and/or (b) a light chain having the amino acid sequence of SEQID NO:221.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:244; aCDR2 sequence consisting of SEQ ID NO:246; and a CDR3 sequenceconsisting of SEQ ID NO:248; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:264; a CDR2 sequenceconsisting of SEQ ID NO:266; and a CDR3 sequence consisting of SEQ IDNO:268. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise (a) a variable heavy chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:242 and/or (b) a variable light chain comprisingan amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO:262. Preferably, the anti-human ACTHantibody or antibody fragment comprises (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO:242, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO:262. Morespecifically, the anti-human ACTH antibody or antibody fragmentcomprises (a) a heavy chain having the amino acid sequence of SEQ IDNO:241, and/or (b) a light chain having the amino acid sequence of SEQID NO:261.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:284; aCDR2 sequence consisting of SEQ ID NO:286; and a CDR3 sequenceconsisting of SEQ ID NO:288; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:304; a CDR2 sequenceconsisting of SEQ ID NO:306; and a CDR3 sequence consisting of SEQ IDNO:308. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:282, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:302. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:282, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:302. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:281, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:301.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:324; aCDR2 sequence consisting of SEQ ID NO:326; and a CDR3 sequenceconsisting of SEQ ID NO:328; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:344; a CDR2 sequenceconsisting of SEQ ID NO:346; and a CDR3 sequence consisting of SEQ IDNO:348. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:322, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:342. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:322, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:342. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:321, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:341.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:364; aCDR2 sequence consisting of SEQ ID NO:366; and a CDR3 sequenceconsisting of SEQ ID NO:368; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:384; a CDR2 sequenceconsisting of SEQ ID NO:386; and a CDR3 sequence consisting of SEQ IDNO:388. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:362, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:382. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:362, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:382. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:361, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:381.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:404; aCDR2 sequence consisting of SEQ ID NO:406; and a CDR3 sequenceconsisting of SEQ ID NO:408; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:424; a CDR2 sequenceconsisting of SEQ ID NO:426; and a CDR3 sequence consisting of SEQ IDNO:428. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:402, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:422. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:402, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:422. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:401, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:421.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:444; aCDR2 sequence consisting of SEQ ID NO:446; and a CDR3 sequenceconsisting of SEQ ID NO:448; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:464; a CDR2 sequenceconsisting of SEQ ID NO:466; and a CDR3 sequence consisting of SEQ IDNO:468. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise (a) a variable heavy chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:442 and/or (b) a variable light chain comprisingan amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO:462. Preferably, the anti-human ACTHantibody or antibody fragment comprises (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO:442, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO:462. Morespecifically, the anti-human ACTH antibody or antibody fragmentcomprises (a) a heavy chain having the amino acid sequence of SEQ IDNO:441, and/or (b) a light chain having the amino acid sequence of SEQID NO:461.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:484; aCDR2 sequence consisting of SEQ ID NO:486; and a CDR3 sequenceconsisting of SEQ ID NO:488; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:504; a CDR2 sequenceconsisting of SEQ ID NO:506; and a CDR3 sequence consisting of SEQ IDNO:508. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:482, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:502. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:482, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:502. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:481, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:501.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:524; aCDR2 sequence consisting of SEQ ID NO:526; and a CDR3 sequenceconsisting of SEQ ID NO:528; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:544; a CDR2 sequenceconsisting of SEQ ID NO:546; and a CDR3 sequence consisting of SEQ IDNO:548. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:522, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:542. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:522, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:542. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:521, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:541.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:564; aCDR2 sequence consisting of SEQ ID NO:566; and a CDR3 sequenceconsisting of SEQ ID NO:568; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:584; a CDR2 sequenceconsisting of SEQ ID NO:586; and a CDR3 sequence consisting of SEQ IDNO:588. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:562, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:582. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:562, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:582. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:561, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:581.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:604; aCDR2 sequence consisting of SEQ ID NO:606; and a CDR3 sequenceconsisting of SEQ ID NO:608; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:624; a CDR2 sequenceconsisting of SEQ ID NO:626; and a CDR3 sequence consisting of SEQ IDNO:628. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise a variable heavy chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:602, and/or a variable light chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:622. Preferably, the anti-human ACTH antibody orantibody fragment comprises (a) a variable heavy chain having the aminoacid sequence of SEQ ID NO:602, and/or (b) a variable light chain havingthe amino acid sequence of SEQ ID NO:622. More specifically, theanti-human ACTH antibody or antibody fragment comprises (a) a heavychain having the amino acid sequence of SEQ ID NO:601, and/or (b) alight chain having the amino acid sequence of SEQ ID NO:621.

In another specific embodiment, the human, humanized or chimerizedanti-human ACTH antibody or antibody fragment comprises (a) a variableheavy chain comprising a CDR1 sequence consisting of SEQ ID NO:644; aCDR2 sequence consisting of SEQ ID NO:646; and a CDR3 sequenceconsisting of SEQ ID NO:648; and/or (b) a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:664; a CDR2 sequenceconsisting of SEQ ID NO:666; and a CDR3 sequence consisting of SEQ IDNO:668. Alternatively, the anti-human ACTH antibody or antibody fragmentmay comprise (a) a variable heavy chain comprising an amino acidsequence with at least 80, 85, 90, 95, 96, 97, 98, or 99% sequenceidentity to SEQ ID NO:642 and/or (b) a variable light chain comprisingan amino acid sequence with at least 80, 85, 90, 95, 96, 97, 98, or 99%sequence identity to SEQ ID NO:662. Preferably, the anti-human ACTHantibody or antibody fragment comprises (a) a variable heavy chainhaving the amino acid sequence of SEQ ID NO:642, and/or (b) a variablelight chain having the amino acid sequence of SEQ ID NO:662. Morespecifically, the anti-human ACTH antibody or antibody fragmentcomprises (a) a heavy chain having the amino acid sequence of SEQ IDNO:641, and/or (b) a light chain having the amino acid sequence of SEQID NO:661.

In one embodiment, the anti-human ACTH antibody or antibody fragmentsare selected from the group consisting of chimeric, humanized, and humanantibodies or antibody fragments, preferably human, humanized orchimerized antibodies or antibody fragments, which may be selected fromthe group consisting of scFvs, camelbodies, nanobodies, IgNAR, Fabfragments, Fab′ fragments, MetMab like antibodies, monovalent antibodyfragments, and F(ab′)₂ fragments.

The anti-human ACTH antibody or antibody fragment may substantially orentirely lack N-glycosylation and/or O-glycosylation. The anti-humanACTH antibody or antibody fragment may comprise a human constant domain,e.g., IgG1, IgG2, IgG3, or IgG4. In one aspect, the anti-human ACTHantibody or antibody fragment comprises an Fc region that has beenmodified to alter at least one of effector function, half-life,proteolysis, or glycosylation. For example, the Fc region may containone or more mutations that alters or eliminates N- and/orO-glycosylation.

In another embodiment, the anti-human ACTH antibody or antibody fragmentis directly or indirectly attached to another moiety, such as adetectable label or therapeutic agent.

In another embodiment, the anti-human ACTH antibody or antibody fragmentinhibits or neutralizes at least one biological effect elicited by ACTHwhen such antibody is administered to a human subject. For example, theantibody or antibody fragment is capable of inhibiting the binding ofACTH to an MCR, i.e., MC1R MC2R, MC3R, MC4R and/or MC5R. Preferably, theanti-human ACTH antibody or antibody fragment neutralizes or inhibitsACTH activation of MC2R; at least one of MC1R, MC2R, MC3R, MC4R andMC5R; at least one of MC2R, MC3R, and MC4R; each of MC2R, MC3R, andMC4R; or each of MC1R, MC2R, MC3R, MC4R and MC5R.

In one embodiment, the anti-human ACTH antibody or antibody fragmentinhibits ACTH-induced cortisol, corticosterone and/or aldosteronesecretion. The anti-human ACTH antibody or antibody fragment, whenadministered to a human subject, can also reduce plasma cortisol,corticosterone, and/or aldosterone levels.

In one embodiment, the anti-human ACTH antibody or antibody fragmentbinds to ACTH with a K_(D) that is less than about 100 nM, less thanabout 10 nM, less than about 1 nM, less than about 100 pM, less thanabout 50 pM, less than about 40 pM, less than about 25 pM, less thanabout 1 pM, between about 10 pM and about 100 pM, between about 1 pM andabout 100 pM, or between about 1 pM and about 10 pM.

Preferably, the anti-human ACTH antibody or antibody fragment hasstronger affinity for ACTH₁₋₃₉ as compared to alpha-MSH or CLIP, i.e.,although there is some cross-reactivity, the antibodies preferentiallybind to ACTH₁₋₃₉ as compared to alpha-MSH or CLIP. For example, theaffinity of said antibody or antibody fragment to ACTH₁₋₃₉ is at least10-fold, 100-fold, 1000-fold or stronger than the affinity of saidantibody or antibody fragment to alpha-MSH or CLIP (e.g., the K_(D) ofsaid antibody or fragment for binding to human ACTH is 10-, 100-, or1000-fold lower than the K_(D) for binding to alpha-MSH or CLIP).

More preferably, for example, the anti-human ACTH antibody or antibodyfragment binds to ACTH₁₋₃₉ but does not bind to alpha-MSH.

In one embodiment, the anti-human ACTH antibody or antibody fragment isattached to at least one effector moiety, e.g., which comprises achemical linker. In another embodiment, the anti-human ACTH antibody orantibody fragment is attached to one or more detectable moieties, e.g.,which comprises a fluorescent dye, enzyme, substrate, bioluminescentmaterial, radioactive material, chemiluminescent moiety, or mixturesthereof.

In one embodiment, the anti-human ACTH antibody or antibody fragment isattached to one or more functional moieties.

The invention also contemplates antibodies, e.g., anti-idiotypicantibodies, produced against an anti-human ACTH antibody or antibodyfragment as described above. Furthermore, the invention provides amethod of using the anti-idiotypic antibody to monitor the in vivolevels of said anti-ACTH antibody or antibody fragment in a subject orto neutralize said anti-ACTH antibody in a subject being administeredsaid anti-ACTH antibody or antibody fragment.

Moreover, the present invention encompasses a composition suitable fortherapeutic, prophylactic, or a diagnostic use comprising atherapeutically, prophylactically or diagnostically effective amount ofat least one anti-human ACTH antibody or antibody fragment as describedherein. The composition may be suitable for subcutaneous administration,intravenous administration, and/or topical administration. Thecomposition may be lyophilized. In some embodiments, the compositionfurther comprises a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative, or mixture thereof. Additionally,in some embodiments, the composition further comprises another activeagent, e.g., selected from the group consisting of ketoconazole(Nizoral®), aminoglutethimide (Cytadren®), metyrapone (Metopirone®),mitotane (Lysodren®) etomidate (Amidate®), cyproheptadine (Periactin® orPeritol®), valproic acid (Depakote®), cabergoline (Dostinex®),pasireotide (Signifor®), rosiglitazone (Avandia®), conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), and satavaptan(SR121463, planned trade name Aquilda®), mifepristone (Korlym®),armodafinil (Nuvigil®) and modafinil (Provigil®). Additionally, in otherembodiments, the composition may be used in conjunction withsupplemental oxygen, continuous positive airway pressure (CPAP), bilevelpositive airway pressure (BPAP), expiratory positive airway pressure(EPAP), adaptive servo-ventilation (ASV), oral appliances,uvulopalatopharyngoplasty (UPPP), maxillomandibular advancement, nasalsurgery, and removal of tonsils and/or adenoids to treat sleep apnea.

In some embodiments, a composition containing the subject antibody mayfurther comprise another active agent, or a therapeutic regimencomprising administration of the subject antibody may includeadministration of at least one other agent. Said other agent or agentsmay be an agent that treats a condition associated with ACTH, such asACTH-driven hypercortisolism, acute coronary syndrome, acute heartfailure, Alzheimer's disease, anxiety disorders, atherosclerosis, atrialfibrillation, cachexia, cancer (such as Cushing's Syndrome resultingfrom ectopic ACTH expression, e.g., in small cell lung cancer, non-smallcell lung cancer (NSCLC), pancreatic carcinoma, neural tumors, orthymoma), cardiac conditions, cardiac fibrosis, cardiovasculardisorders, chronic renal failure, chronic stress syndrome, cognitivedysfunction, congestive heart failure, Conn's syndrome, coronary heartdiseases, Cushing's Disease, Cushing's Syndrome, depression, diabetes,endothelial dysfunction, exercise intolerance, familialhyperaldosteronism, fibrosis, galactorrhea, heart failure,hyperaldosteronism, hypercortisolemia, hypertension, hyperinsulinemia,hypokalemia, impaired cardiac function, increased formation of collagen,inflammation, metabolic syndrome, muscle atrophy, conditions associatedwith muscle atrophy, myocardiac fibrosis, nephropathy, obesity,post-myocardial infarction, primary hyperaldosteronism, remodelingfollowing hypertension, renal failure, restenosis, secondaryhyperaldosteronism, sleep apnea, stress related conditions, or syndromeX, or a condition that may co-present with one or more of saidconditions, such as hypercholesterolemia. Said additional agent oragents may include without limitation thereto one or more of: Accupril(quinapril), Aceon (perindopril), Adalat, Adalat CC, Aldactone(spironolactone), aldosterone receptor blockers, alpha-adrenergicreceptor blockers, alpha-glucosidase inhibitors, Altace (ramipril),Alteplase, aminoglutethimide (Cytadren®), amiodarone, angiotensinconverting enzyme (ACE) Inhibitors, angiotensin II receptor antagonists,Angiotensin II receptor blockers (ARBs), antiarrhythmics,anti-cholesterol drugs, anti-clotting agents, antidiabetogenic drugs,anti-hypertensive agents, antiplatelet drugs, ApoA-1 mimics, aspirin,Atacand (candesartan), Avapro (irbesartan), beta blockers,beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR,cholesteryl ester transfer protein (CETP) inhibitors, conivaptan(Vaprisol®), Cordarone (amiodarone), Coreg (carvedilol), Covera-HS,Cozaar (losartan), cyproheptadine (Periactin® or Peritol®), Demadex(torsemide), digoxin, Dilacor XR, Dilatrate-SR, Diltia XT, Diovan(valsartan), dipeptidyl peptidase-4 inhibitors, diuretics, Dobutrex(dobutamine), drugs that suppress ACTH secretion, drugs that suppresscortisol secretion, dual angiotensin converting enzyme/neutralendopeptidase (ACE/NEP) inhibitors, endothelin antagonists, endothelinreceptor blockers, Esidrix (hydrochlorothiazide), etomidate (Amidate®),Fragmin, gemfibrozil (Lopid, Gemcor), glucocorticoid receptorantagonists, heart failure drugs, Heparin, HMG-Co-A reductaseinhibitors, holestyramine (Questran), IMDUR (isosorbide mononitrate),Inderal (propranolol), inhibitors of a Na—K-ATPase membrane pump,inhibitors of steroidogenesis, insulin therapies, Iso-Bid, Isonate,Isoptin, Isoptin SR, Isorbid (isosorbide dinitrate), Isordil, Isotrate,ketoconazole (Nizoral®), Lasix (furosemide), lixivaptan (VPA-985),Lopressor, Lotensin (benazepril), Lovenox, Mavik (trandolapril),meglitinides, metyrapone (Metopirone®), Micardis (telmisartan),mifepristone (Korlym®), mitotane (Lysodren®), Monopril (fosinopril),neutral endopeptidase (NEP) inhibitors, Normodyne, Norvasc (amlodipine),obesity-reducing agents, Omacor, pantethine, pasireotide (Signifor®),Plendil (felodipine), PPAR-gamma agonists, Primacor (milrinone),Prinivil, Procanbid (procainamide), Procardia, Procardia XL(nifedipine), renin inhibitors, Reteplase, rosiglitazone (Avandia®),satavaptan (SR121463, planned trade name Aquilda®), Sectral(acebutolol), somatostatin analogs, Sorbitrate (isosorbide dinitrate),statins, Streptokinase, Sular (nisoldipine), sulfonylurea, Tambocor(flecainide), Tenecteplase, Tenormin (atenolol), thiazolidinediones,Tiazac (diltiazem), Tissue plasminogen activator (TPA), tolvaptan(OPC-41061), Toprol-XL (metoprolol), Trandate (labetalol), Univasc(moexipril), Urokinase, valproic acid (Depakote®), vaptans, Vascor(bepridil), vasodilators, Vasodilators, vasopressin antagonists, Vasotec(enalapril), Verelan, Verelan PM (verapamil), warfarin (Coumadin),Zaroxolyn (metolazone), Zebeta (bisoprolol), or Zestril (lisinopril).Further exemplary active agents include one or more corticosteroids,including glucocorticoids and/or mineralocorticoids (including agentshaving one or both of glucocorticoid and/or mineralocorticoid activity),such as cortisol (hydrocortisone), cortisone, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone (e.g., fludrocortisone acetate),deoxycorticosterone (e.g., deoxycorticosterone acetate (DOCA)), and/oraldosterone.

The present invention further contemplates an isolated nucleic acidsequence or nucleic acid sequences encoding an anti-human ACTH antibodyor antibody fragment described herein as well as a vector or vectorscontaining these isolated nucleic acid sequence or sequences.Additionally, the invention provides a host cell comprising theseisolated nucleic acid sequence or sequences or the vector or set forthabove. The host cell may be a mammalian, bacterial, fungal, yeast, avianor insect cell. Preferably, the host cell is a filamentous fungi or ayeast. More preferably, the yeast is selected from the from thefollowing genera: Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces;Dekkera; Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces;Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Morepreferably, the yeast species is of the genus Pichia. Most preferably,the species of Pichia is selected from Pichia pastoris, Pichiamethanolica and Hansenula polymorpha (Pichia angusta).

The invention further provides a method of expressing an anti-human ACTHantibody or antibody fragment, typically a human, humanized, or chimericantibody or antibody fragment, the method comprising culturing the hostcell described herein under conditions that provide for expression ofsaid antibody or antibody fragment. The host cell may be a polyploidyeast culture that stably expresses and secretes into the culture mediumat least 10-25 mg/liter of said antibody or antibody fragment. Thepolyploid yeast may be made by a method that comprises: (i) introducingat least one expression vector containing one or more heterologouspolynucleotides encoding said antibody operably linked to a promoter anda signal sequence into a haploid yeast cell; (ii) producing by mating orspheroplast fusion a polyploid yeast from said first and/or secondhaploid yeast cell; (iii) selecting polyploid yeast cells that stablyexpress said antibody; and (iv) producing stable polyploid yeastcultures from said polyploid yeast cells that stably express saidantibody into the culture medium. Preferably, the yeast species is ofthe genus Pichia.

The invention further relates to the therapeutic and diagnostic uses ofanti-ACTH antibodies and antibody fragments. In one embodiment, theinvention provides a method for blocking, inhibiting or neutralizing oneor more biological effects associated with ACTH and/or treating anycondition associated with elevated cortisol levels comprisingadministering to a subject in need thereof an effective amount of ananti-human adrenocorticotrophic hormone (“ACTH”) antibody or antibodyfragment. Also, the invention provides a method for treating orpreventing a condition associated with elevated ACTH levels in asubject, comprising administering to a subject in need thereof aneffective amount of an anti-human adrenocorticotrophic hormone (“ACTH”)antibody or antibody fragment. Exemplary conditions include, but are notlimited to, ACTH-driven hypercortisolism (Cushing's Disease and/orCushing's Syndrome), obesity, diabetes, sleep apnea, depression, anxietydisorders, cancer (such as Cushing's Syndrome resulting from ectopicACTH expression, e.g., in small cell lung cancer, non-small cell lungcancer (NSCLC), pancreatic carcinoma, neural tumors, or thymoma), muscleatrophies, hypertension, cognitive dysfunction, galactorrhea, metabolicsyndromes, and hyperaldosteronism including primary hyperaldosteronism(such as Conn's syndrome), secondary hyperaldosteronism, familialhyperaldosteronism, and other conditions associated with ACTH describedherein.

The invention further provides a method for neutralizing ACTH-inducedMCR signaling, comprising administering to a subject in need thereof aneffective amount of an anti-human adrenocorticotrophic hormone (“ACTH”)antibody or antibody fragment. Moreover, the invention encompasses amethod for inhibiting ACTH-induced cortisol, corticosterone, and/oraldosterone secretion, comprising administering to a subject in needthereof an effective amount of an anti-human adrenocorticotrophichormone (“ACTH”) antibody or antibody fragment. Furthermore, theinvention contemplates a method for reducing ACTH-induced plasmacortisol, corticosterone, and/or aldosterone levels in a subject in needthereof, comprising administering to a subject in need thereof aneffective amount of an anti-human adrenocorticotrophic hormone (“ACTH”)antibody or antibody fragment.

In these methods, the anti-human ACTH antibody or antibody fragmentpreferably does not substantially interact with (bind) a polypeptideconsisting of: (i) the 13 N-terminal amino acid residues of ACTH(ACTH₁₋₁₃) and/or alpha-MSH, or (ii) the 22 C-terminal amino acidresidues of ACTH (ACTH₁₈₋₃₉).

In exemplary embodiments in these methods, the anti-human ACTH antibodyor antibody fragment, preferably a human, humanized or chimerizedanti-ACTH antibody or antibody fragment binds to the same linear orconformational epitope(s) and/or competes for binding to the same linearor conformational epitope(s) on human ACTH as an anti-human ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H, preferably Ab13.H and preferably theat least one isolated anti-human ACTH antibody or antibody fragmentinhibits ACTH-induced signaling via a MCR, e.g., an MCR is selected fromthe group consisting of MC1R, MC2R, MC3R, MC4R and MC5R.

In exemplary embodiments the epitope(s) bound by the administeredanti-human ACTH antibody or antibody fragment is identified using abinding assay that detects the binding of said anti-human ACTH antibodyor antibody fragment to one or more peptides in a library of overlappinglinear peptide fragments that span the full length of human ACTH.

In exemplary embodiments, the methods will use anti-human ACTHantibodies or antibody fragments contain at least 2 complementaritydetermining regions (CDRs) of an anti-human ACTH antibody selected fromthe group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H, preferably Ab13.H. In exemplary embodiments, the antibody orfragment will retain the V_(H) CDR3 and/or the V_(L) CDR3 of one ofAb13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H,Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, OR Ab17.H, preferablyAb13.H.

In exemplary embodiments, the methods will use anti-ACTH antibodies orantibody fragments contain at least 3 CDRs of an anti-ACTH antibodyselected from the group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H,Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H,Ab13.H, Ab15.H, and Ab17.H, preferably Ab13.H.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments contain at least 4 CDRs of an anti-ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H, preferably Ab13.H.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments contain at least 5 CDRs of an anti-ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H, preferably Ab13.H.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments contain all 6 CDRs of an anti-ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H, preferably Ab13.H.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:4; a CDR2sequence consisting of SEQ ID NO:6; and a CDR3 sequence consisting ofSEQ ID NO:8; and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:24; a CDR2 sequence consisting of SEQ ID NO:26;and a CDR3 sequence consisting of SEQ ID NO:28; (b) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO:2; and/or a variablelight chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:22; (c) a variableheavy chain having the amino acid sequence of SEQ ID NO:2; and/or avariable light chain having the amino acid sequence of SEQ ID NO:22; or(d) a heavy chain having the amino acid sequence of SEQ ID NO: 1, and/ora light chain having the amino acid sequence of SEQ ID NO:21.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:44; a CDR2sequence consisting of SEQ ID NO:46; and a CDR3 sequence consisting ofSEQ ID NO:48, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:64; a CDR2 sequence consisting of SEQ ID NO:66;and a CDR3 sequence consisting of SEQ ID NO:68; (b) a variable heavychain comprising an amino acid sequence with at least 80, 85, 90, 95,96, 97, 98, or 99% sequence identity to SEQ ID NO:42, and/or a variablelight chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:62; (c) a variableheavy chain having the amino acid sequence of SEQ ID NO:42, and/or avariable light chain having the amino acid sequence of SEQ ID NO:62; or(d) a heavy chain having the amino acid sequence of SEQ ID NO:41, and/ora light chain having the amino acid sequence of SEQ ID NO:61.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody that comprise (a) a variable heavy chaincomprising a CDR1 sequence consisting of SEQ ID NO:84; a CDR2 sequenceconsisting of SEQ ID NO:86; and a CDR3 sequence consisting of SEQ IDNO:88, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 104; a CDR2 sequence consisting of SEQ ID NO:106; and a CDR3 sequence consisting of SEQ ID NO: 108; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:82, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 102; (c)a variable heavy chain having the amino acid sequence of SEQ ID NO:82,and/or a variable light chain having the amino acid sequence of SEQ IDNO:102; or (d) a heavy chain having the amino acid sequence of SEQ IDNO:81, and/or a light chain having the amino acid sequence of SEQ IDNO:101.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO: 124; a CDR2sequence consisting of SEQ ID NO: 126 and a CDR3 sequence consisting ofSEQ ID NO: 128, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 144; a CDR2 sequence consisting of SEQ ID NO:146; and a CDR3 sequence consisting of SEQ ID NO: 148; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 122 and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 142; (c)a variable heavy chain having the amino acid sequence of SEQ ID NO: 122,and/or a variable light chain having the amino acid sequence of SEQ IDNO: 142; or (d) a heavy chain having the amino acid sequence of SEQ IDNO: 121, and/or a light chain having the amino acid sequence of SEQ IDNO: 141.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO: 164; a CDR2sequence consisting of SEQ ID NO: 166; and a CDR3 sequence consisting ofSEQ ID NO: 168, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO: 184; a CDR2 sequence consisting of SEQ ID NO:186; and a CDR3 sequence consisting of SEQ ID NO: 188; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 162, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 182; (c)a variable heavy chain having the amino acid sequence of SEQ ID NO: 162,and/or a variable light chain having the amino acid sequence of SEQ IDNO:182; or (d) a heavy chain having the amino acid sequence of SEQ IDNO:161, and/or a light chain having the amino acid sequence of SEQ IDNO:181.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:204; a CDR2sequence consisting of SEQ ID NO:206; and a CDR3 sequence consisting ofSEQ ID NO:208, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:224; a CDR2 sequence consisting of SEQ IDNO:226; and a CDR3 sequence consisting of SEQ ID NO:228; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:202 and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:222; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:202,and/or a variable light chain having the amino acid sequence of SEQ IDNO:222; or (d) a heavy chain having the amino acid sequence of SEQ IDNO:201, and/or a light chain having the amino acid sequence of SEQ IDNO:221.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:244; a CDR2sequence consisting of SEQ ID NO:246; and a CDR3 sequence consisting ofSEQ ID NO:248, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:264; a CDR2 sequence consisting of SEQ IDNO:266; and a CDR3 sequence consisting of SEQ ID NO:268; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:242, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:262; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:242,and/or a variable light chain having the amino acid sequence of SEQ IDNO:262; (d) a heavy chain having the amino acid sequence of SEQ IDNO:241, and/or a light chain having the amino acid sequence of SEQ IDNO:261.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:284; a CDR2sequence consisting of SEQ ID NO:286; and a CDR3 sequence consisting ofSEQ ID NO:288, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:304; a CDR2 sequence consisting of SEQ IDNO:306; and a CDR3 sequence consisting of SEQ ID NO:308; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:282, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:302; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:282,and/or a variable light chain having the amino acid sequence of SEQ IDNO:302; (d) a heavy chain having the amino acid sequence of SEQ IDNO:281, and/or a light chain having the amino acid sequence of SEQ IDNO:301.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:324; a CDR2sequence consisting of SEQ ID NO:326; and a CDR3 sequence consisting ofSEQ ID NO:328, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:344; a CDR2 sequence consisting of SEQ IDNO:346; and a CDR3 sequence consisting of SEQ ID NO:348; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:322, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:342; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:322,and/or a variable light chain having the amino acid sequence of SEQ IDNO:342; (d) a heavy chain having the amino acid sequence of SEQ IDNO:321, and/or a light chain having the amino acid sequence of SEQ IDNO:341.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:364; a CDR2sequence consisting of SEQ ID NO:366; and a CDR3 sequence consisting ofSEQ ID NO:368, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:384; a CDR2 sequence consisting of SEQ IDNO:386; and a CDR3 sequence consisting of SEQ ID NO:388; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:362, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:382; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:362,and/or a variable light chain having the amino acid sequence of SEQ IDNO:382; (d) a heavy chain having the amino acid sequence of SEQ IDNO:361, and/or a light chain having the amino acid sequence of SEQ IDNO:381.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:404; a CDR2sequence consisting of SEQ ID NO:406; and a CDR3 sequence consisting ofSEQ ID NO:408, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:424; a CDR2 sequence consisting of SEQ IDNO:426; and a CDR3 sequence consisting of SEQ ID NO:428; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:402, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:422; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:402,and/or a variable light chain having the amino acid sequence of SEQ IDNO:422; (d) a heavy chain having the amino acid sequence of SEQ IDNO:401, and/or a light chain having the amino acid sequence of SEQ IDNO:421.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:444; a CDR2sequence consisting of SEQ ID NO:446; and a CDR3 sequence consisting ofSEQ ID NO:448, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:464; a CDR2 sequence consisting of SEQ IDNO:466; and a CDR3 sequence consisting of SEQ ID NO:468; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:442, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:462; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:442,and/or a variable light chain having the amino acid sequence of SEQ IDNO:462; (d) a heavy chain having the amino acid sequence of SEQ IDNO:441, and/or a light chain having the amino acid sequence of SEQ IDNO:461.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:484; a CDR2sequence consisting of SEQ ID NO:486; and a CDR3 sequence consisting ofSEQ ID NO:488, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:504; a CDR2 sequence consisting of SEQ IDNO:506; and a CDR3 sequence consisting of SEQ ID NO:508; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:482, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:502; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:482,and/or a variable light chain having the amino acid sequence of SEQ IDNO:502; (d) a heavy chain having the amino acid sequence of SEQ IDNO:481, and/or a light chain having the amino acid sequence of SEQ IDNO:501.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:524; a CDR2sequence consisting of SEQ ID NO:526; and a CDR3 sequence consisting ofSEQ ID NO:528, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:544; a CDR2 sequence consisting of SEQ IDNO:546; and a CDR3 sequence consisting of SEQ ID NO:548; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:522, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:542; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:522,and/or a variable light chain having the amino acid sequence of SEQ IDNO:542; (d) a heavy chain having the amino acid sequence of SEQ IDNO:521, and/or a light chain having the amino acid sequence of SEQ IDNO:541.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:564; a CDR2sequence consisting of SEQ ID NO:566; and a CDR3 sequence consisting ofSEQ ID NO:568, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:584; a CDR2 sequence consisting of SEQ IDNO:586; and a CDR3 sequence consisting of SEQ ID NO:588; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:562, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:582; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:562,and/or a variable light chain having the amino acid sequence of SEQ IDNO:582; (d) a heavy chain having the amino acid sequence of SEQ IDNO:561, and/or a light chain having the amino acid sequence of SEQ IDNO:581.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:604; a CDR2sequence consisting of SEQ ID NO:606; and a CDR3 sequence consisting ofSEQ ID NO:608, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:624; a CDR2 sequence consisting of SEQ IDNO:626; and a CDR3 sequence consisting of SEQ ID NO:628; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:602, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:622; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:602,and/or a variable light chain having the amino acid sequence of SEQ IDNO:622; (d) a heavy chain having the amino acid sequence of SEQ IDNO:601, and/or a light chain having the amino acid sequence of SEQ IDNO:621.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise (a) a variable heavychain comprising a CDR1 sequence consisting of SEQ ID NO:644; a CDR2sequence consisting of SEQ ID NO:646; and a CDR3 sequence consisting ofSEQ ID NO:648, and/or a variable light chain comprising a CDR1 sequenceconsisting of SEQ ID NO:664; a CDR2 sequence consisting of SEQ IDNO:666; and a CDR3 sequence consisting of SEQ ID NO:668; (b) a variableheavy chain comprising an amino acid sequence with at least 80, 85, 90,95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:642, and/or avariable light chain comprising an amino acid sequence with at least 80,85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:662; (c) avariable heavy chain having the amino acid sequence of SEQ ID NO:642,and/or a variable light chain having the amino acid sequence of SEQ IDNO:662; (d) a heavy chain having the amino acid sequence of SEQ IDNO:641, and/or a light chain having the amino acid sequence of SEQ IDNO:661.

In other exemplary embodiments, the anti-ACTH antibodies or antibodyfragments used in the methods are chimeric, humanized, and humanantibodies or antibody fragments.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments selected from the group consisting ofscFvs, camelbodies, nanobodies, IgNAR, Fab fragments, Fab′ fragments,MetMab like antibodies, monovalent antibody fragments, and F(ab′)₂fragments.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that substantially or entirely lackN-glycosylation and/or O-glycosylation.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise a human constant domain,e.g., an IgG1, IgG2, IgG3, or IgG4 antibody.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that comprise an Fc region that hasbeen modified to alter at least one of effector function, half-life,proteolysis, or glycosylation.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments having an Fc region which contains oneor more mutations that alters or eliminates N- and/or O-glycosylation.

In other exemplary embodiments, the methods will use a human orhumanized anti-ACTH antibody or antibody fragment.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that bind to ACTH with a K_(D) of lessthan or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M,5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that bind to ACTH with a K_(D) of lessthan or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, or10⁻¹² M.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that bind to ACTH with an off-rate(k_(d)) of less than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻⁵ s⁻¹, or10⁻⁵ s⁻¹.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that are directly or indirectlyattached to a therapeutic agent.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that are attached to one or moredetectable moieties.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments comprising a detectable moiety, e.g.,that comprises a fluorescent dye, enzyme, substrate, bioluminescentmaterial, radioactive material, chemiluminescent moiety, or mixturesthereof.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that are attached to one or morefunctional moieties.

In other exemplary embodiments, the methods will use anti-ACTHantibodies or antibody fragments that reduce plasma cortisol,corticosterone, and/or aldosterone levels.

In other exemplary embodiments, the methods further compriseadministering separately or co-administering another agent, e.g.,selected from the group consisting of ketoconazole (Nizoral®),aminoglutethimide (Cytadren®), metyrapone (Metopirone®), mitotane(Lysodren®) etomidate (Amidate®), cyproheptadine (Periactin® orPeritol®), valproic acid (Depakote®), cabergoline (Dostinex®),pasireotide (Signifor®), rosiglitazone (Avandia®), conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), and satavaptan(SR121463, planned trade name Aquilda®), mifepristone (Korlym®),armodafinil (Nuvigil®) and modafinil (Provigil®). Further, saidadditional agent may include without limitation thereto one or more of:Accupril (quinapril), Aceon (perindopril), Adalat, Adalat CC, Aldactone(spironolactone), aldosterone receptor blockers, alpha-adrenergicreceptor blockers, alpha-glucosidase inhibitors, Altace (ramipril),Alteplase, aminoglutethimide (Cytadren®), amiodarone, angiotensinconverting enzyme (ACE) Inhibitors, angiotensin II receptor antagonists,Angiotensin II receptor blockers (ARBs), antiarrhythmics,anti-cholesterol drugs, anti-clotting agents, antidiabetogenic drugs,anti-hypertensive agents, antiplatelet drugs, ApoA-1 mimics, aspirin,Atacand (candesartan), Avapro (irbesartan), beta blockers,beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR, CETPinhibitors, conivaptan (Vaprisol®), Cordarone (amiodarone), Coreg(carvedilol), Covera-HS, Cozaar (losartan), cyproheptadine (Periactin®or Peritol®), Demadex (torsemide), digoxin, Dilacor XR, Dilatrate-SR,Diltia XT, Diovan (valsartan), dipeptidyl peptidase-4 inhibitors,diuretics, Dobutrex (dobutamine), drugs that suppress ACTH secretion,drugs that suppress cortisol secretion, dual angiotensin convertingenzyme/neutral endopeptidase (ACE/NEP) inhibitors, endothelinantagonists, endothelin receptor blockers, Esidrix(hydrochlorothiazide), etomidate (Amidate®), Fragmin, gemfibrozil(Lopid, Gemcor), glucocorticoid receptor antagonists, heart failuredrugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine (Questran),IMDUR (isosorbide mononitrate), Inderal (propranolol), inhibitors of aNa—K-ATPase membrane pump, inhibitors of steroidogenesis, insulintherapies, Iso-Bid, Isonate, Isoptin, Isoptin SR, Isorbid (isosorbidedinitrate), Isordil, Isotrate, ketoconazole (Nizoral®), Lasix(furosemide), lixivaptan (VPA-985), Lopressor, Lotensin (benazepril),Lovenox, Mavik (trandolapril), meglitinides, metyrapone (Metopirone®),Micardis (telmisartan), mifepristone (Korlym®), mitotane (Lysodren®),Monopril (fosinopril), neutral endopeptidase (NEP) inhibitors,Normodyne, Norvasc (amlodipine), obesity-reducing agents, Omacor,pantethine, pasireotide (Signifor®), Plendil (felodipine), PPAR-gammaagonists, Primacor (milrinone), Prinivil, Procanbid (procainamide),Procardia, Procardia XL (nifedipine), renin inhibitors, Reteplase,rosiglitazone (Avandia®), satavaptan (SR121463, planned trade nameAquilda®), Sectral (acebutolol), somatostatin analogs, Sorbitrate(isosorbide dinitrate), statins, Streptokinase, Sular (nisoldipine),sulfonylurea, Tambocor (flecainide), Tenecteplase, Tenormin (atenolol),thiazolidinediones, Tiazac (diltiazem), Tissue plasminogen activator(TPA), tolvaptan (OPC-41061), Toprol-XL (metoprolol), Trandate(labetalol), Univasc (moexipril), Urokinase, valproic acid (Depakote®),vaptans, Vascor (bepridil), vasodilators, Vasodilators, vasopressinantagonists, Vasotec (enalapril), Verelan, Verelan PM (verapamil),warfarin (Coumadin), Zaroxolyn (metolazone), Zebeta (bisoprolol), orZestril (lisinopril). Further exemplary active agents include one ormore corticosteroids, including glucocorticoids and/ormineralocorticoids (including agents having one or both ofglucocorticoid and/or mineralocorticoid activity), such as cortisol(hydrocortisone), cortisone, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone (e.g., fludrocortisone acetate),deoxycorticosterone (e.g., deoxycorticosterone acetate (DOCA)), and/oraldosterone. The antibody or antibody fragment or the compositioncontaining the antibody of antibody fragment and the at least one otheragent may be administered concurrently sequentially, e.g., the antibodyor antibody fragment is administered before or after the at least oneother agent.

In yet other exemplary embodiments, the methods further comprise usingthe anti-ACTH antibodies or antibody fragments disclosed herein incombination with supplemental oxygen, continuous positive airwaypressure (CPAP), bilevel positive airway pressure (BPAP), expiratorypositive airway pressure (EPAP), adaptive servo-ventilation (ASV), oralapplicanes, uvulopalatopharyngoplasty (UPPP), maxilloandibularadvancement, nasal surgery, and removal of tonsils and/or adenoids totreat sleep apnea.

In other exemplary methods, the anti-ACTH antibody or antibody fragmentis a human, humanized or chimerized anti-ACTH antibody or antibodyfragment which substantially does not interact with (bind) a polypeptideconsisting of: (i) the 13 N-terminal amino acid residues of ACTH(ACTH₁₋₁₃) and/or alpha-MSH, or (ii) the 22 C-terminal amino acidresidues of ACTH (ACTH₁₈₋₃₉) (Corticotrophin-Like Intermediate peptideor “CLIP”).

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-ACTH antibody orantibody fragment which binds to ACTH₁₋₃₉ with a binding affinity(K_(D)) at least 10-fold, 100-fold, 1000-fold or 10,000-fold strongerthan the binding affinity of said antibody or antibody fragment to (i)ACTH₁₋₁₃ and/or alpha-MSH, and/or (ii) CLIP (i.e., a numerically lowerK_(D) for ACTH₁₋₃₉ by at least 10-fold, 100-fold, 1000-fold or10,000-fold relative to the K_(D) for ACTH₁₋₁₃ and/or alpha-MSH and/orCLIP).

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-human ACTH antibody orantibody fragment which neutralizes or inhibits ACTH activation of MC2R.

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-human ACTH antibody orantibody fragment which neutralizes or inhibits ACTH activation of atleast one of MC2R, MC3R and MC4R.

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-human ACTH antibody orantibody fragment, which neutralizes or inhibits ACTH activation of eachof MC2R, MC3R and MC4R.

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-human ACTH antibody orantibody fragment, which inhibits ACTH-induced corticosterone secretion.

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-human ACTH antibody orantibody fragment, which when administered to a human subject reducesplasma cortisol, corticosterone and/or aldosterone levels.

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-human ACTH antibody orantibody fragment capable of inhibiting the binding of ACTH to a MCR.

In other exemplary embodiments, the anti-ACTH antibody or antibodyfragment is a human, humanized or chimerized anti-human ACTH antibody orantibody fragment, capable of inhibiting the binding of ACTH to at leastone of MC1R, MC2R, MC3R, MC4R and MC5R; at least one of MC2R, MC3R, andMC4R; each of MC2R, MC3R, and MC4R; or each of MC1R, MC2R, MC3R, MC4Rand MC5R.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1G provides the polypeptide sequences of the full-length heavychain for antibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H (SEQ ID NOs: 1; 41; 81; 121; 161; 201; 241; 281; 321; 361;401; 441; 481; 521; 561; 601; 641 respectively) aligned by theirframework regions (FR) and complementarity determining regions (CDRs),and constant regions.

FIG. 2A-2D provide the polypeptide sequences of the full-length lightchain for antibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H (SEQ ID NOs: 21; 61; 101; 141; 181; 221; 261; 301; 341; 381;421; 461; 501; 541; 581; 621; and 661, respectively) aligned by theirframework regions (FR), complementarity determining regions (CDRs), andconstant regions.

FIG. 3A-3S provide the polynucleotide sequences encoding the full-lengthheavy chain for antibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H (SEQ ID NOs: 11; 51; 91; 131; 171; 211; 251; 291; 331; 371;411; 451; 491; 531; 571; 611; and 651, respectively) aligned by theirframework regions (FR), complementarity determining regions (CDRs), andconstant regions.

FIG. 4A-I provide the polynucleotide sequences encoding the full-lengthlight chain for antibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H (SEQ ID NOs: 31; 71; 111; 151; 191; 231; 271; 311; 351; 391;431; 471; 511; 551; 591; 631; and 671 respectively) aligned by theirframework regions (FR), complementarity determining regions (CDRs), andconstant regions.

FIG. 5A provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the variableregion and complementarity determining regions (CDRs) of the heavy chainfor antibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H.

FIG. 5B provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the variableregion and complementarity determining regions (CDRs) of the heavy chainfor antibodies Ab1-Ab7 and Ab9-Ab12.

FIG. 6A provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the constantregion and framework regions (FR) of the heavy chain for antibodiesAb13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H,Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

FIG. 6B provides the polypeptide sequence coordinates for certainantibody heavy chain protein sequence features including the constantregion and framework regions (FR) of the heavy chain for antibodiesAb1-Ab7 and Ab9-Ab12.

FIG. 7A provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the variableregion and complementarity determining regions (CDRs) of the light chainfor antibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H.

FIG. 7B provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the variableregion and complementarity determining regions (CDRs) of the light chainfor antibodies Ab1-Ab7 and Ab9-Ab12.

FIG. 8A provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the constantregion and framework regions (FR) of the light chain for antibodiesAb13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H,Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

FIG. 8B provides the polypeptide sequence coordinates for certainantibody light chain protein sequence features including the constantregion and framework regions (FR) of the light chain for antibodiesAb1-Ab7 and Ab9-Ab12.

FIG. 9A provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the variable regionand complementarity determining regions (CDRs) of the heavy chain forantibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H,Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

FIG. 9B provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the variable regionand complementarity determining regions (CDRs) of the heavy chain forantibodies Ab1-Ab7 and Ab9-Ab12.

FIG. 10A provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the constant regionand framework regions (FR) of the heavy chain for antibodies Ab13, Ab15,Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H,Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

FIG. 10B provides the polynucleotide sequence coordinates for certainantibody heavy chain DNA sequence features including the constant regionand framework regions (FR) of the heavy chain for antibodies Ab1-Ab7 andAb9-Ab12.

FIG. 11A provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the variable regionand complementarity determining regions (CDRs) of the light chain forantibodies Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H,Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

FIG. 11B provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the variable regionand complementarity determining regions (CDRs) of the light chain forantibodies Ab1-Ab7 and Ab9-Ab12.

FIG. 12A provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the constant regionand framework regions (FR) of the light chain for antibodies Ab13, Ab15,Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H,Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

FIG. 12B provides the polynucleotide sequence coordinates for certainantibody light chain DNA sequence features including the constant regionand framework regions (FR) of the light chain for antibodies Ab1-Ab7 andAb9-Ab12.

FIG. 13 provides representative binding data for the subject anti-humanACTH antibodies to human ACTH (specifically, for Ab1).

FIG. 14 provides representative binding data for the subject anti-humanACTH antibodies to human ACTH1-13 and ACTH 18-39 (specifically, forAb1).

FIG. 15 provides representative binding data for the subject anti-humanACTH antibodies to ACTH 1-39 and the inability of human ACTH 1-13 andACTH 18-39 to compete with binding of ACTH 1-39 (specifically, for Ab5).

FIG. 16 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab1) inhibited ACTH-induced cAMP productionin cells expressing MC2R.

FIG. 17 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab5) inhibited ACTH-induced cAMP productionin cells expressing MC2R.

FIG. 18 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab1) inhibited ACTH-induced cAMP productionin cells expressing MC1R.

FIG. 19 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab1) inhibited ACTH-induced cAMP productionin cells expressing MC3R.

FIG. 20 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab1) inhibited ACTH-induced cAMP productionin cells expressing MC4R.

FIG. 21 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab1) inhibited ACTH-induced cAMP productionin cells expressing MC5R.

FIG. 22 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab1) inhibited ACTH-induced cortisolproduction by Y1 cells.

FIG. 23 shows plasma corticosterone levels pre-dose of Ab2 or Ab3 forthe experiments described in Example 6.

FIG. 24 shows plasma corticosterone levels 48 hours after the first doseof Ab2, Ab3, or vehicle control (AD26-10) antibody for the experimentsdescribed in Example 6.

FIG. 25 shows plasma corticosterone levels 48 hours after the seconddose of Ab2, Ab3, or vehicle control (AD26-10) antibody for theexperiments described in Example 6.

FIG. 26 shows plasma corticosterone levels 120 hours after the seconddose of Ab2, Ab3, or vehicle control (AD26-10) antibody for theexperiments described in Example 6.

FIG. 27 shows the percent change in animal weight for animals treatedwith Ab6 and dosed with ACTH using an infusion pump for the experimentsdescribed in Example 7. ANOVA analysis was performed at day 8 to compareVehicle/control antibody (AD26-10) to ACTH/control antibody (AD26-10)which showed a significant difference (p<0.0001), and to compareACTH/Ab6 to ACTH/AD26-10 which also showed a significant difference(p<0.0001).

FIG. 28 shows plasma corticosterone levels before initiation of ACTHdosing and antibody administration for the experiments described inExample 7.

FIG. 29 shows plasma corticosterone levels 24 hours post initiation ofACTH dosing and pre-Ab dose for the experiments described in Example 7.

FIG. 30 shows plasma corticosterone levels 48 hours post initiation ofACTH dosing and 24 hours post Ab dose (Ab6) for the experimentsdescribed in Example 7.

FIG. 31 shows plasma corticosterone levels 96 hours post initiation ofACTH dosing and 72 hours post Ab dose (Ab6) for the experimentsdescribed in Example 7.

FIG. 32 shows plasma corticosterone levels 144 hours post initiation ofACTH dosing and 120 hours post Ab dose (Ab6) for the experimentsdescribed in Example 7.

FIG. 33 shows plasma corticosterone levels 168 hours post initiation ofACTH dosing and 144 hours post Ab dose (Ab6) for the experimentsdescribed in Example 7.

FIG. 34 shows plasma aldosterone levels before the initiation of ACTHdosing and antibody administration for the experiments described inExample 7.

FIG. 35 shows plasma aldosterone levels 24 hours post initiation of ACTHdosing and pre-Ab dose for the experiments described in Example 7.

FIG. 36 shows plasma aldosterone levels 48 hours post initiation of ACTHdosing and 24 hours post Ab dose (Ab6) for the experiments described inExample 7.

FIG. 37 shows plasma aldosterone levels 96 hours post initiation of ACTHdosing and 72 hours post Ab dose (Ab6) for the experiments described inExample 7.

FIG. 38 shows plasma aldosterone levels 144 hours post initiation ofACTH dosing and 120 hours post Ab dose (Ab6) for the experimentsdescribed in Example 7.

FIG. 39 shows plasma aldosterone levels 168 hours post initiation ofACTH dosing and 144 hours post Ab dose (Ab6) for the experimentsdescribed in Example 7.

FIG. 40A-O shows results of binding kinetics measurements for binding ofanti-ACTH antibodies to alanine scanning mutants of human ACTH. Eachupper panel shows results for wild-type huACTH and alanine scanningmutants that were determined to substantially affect binding, indicatingthat these positions formed part of the epitope bound by this antibody.Each lower panel shows traces for all of the remaining alanine scanningmutants (along with wild-type huACTH shown for reference).

FIG. 41 shows the results of alanine scanning mutagenesis used toidentify positions in ACTH that form the epitope bound by each testedantibody. In the column under each antibody name are listed the mutationof which substantially altered the binding kinetics of the antibody toACTH, which was interpreted to indicate that the position forms part ofthe epitope bound by that antibody. For visual illustration thepositions are listed in order of their position, e.g., the seventh rowbelow the header is labeled “7A” for those antibodies for which the 7Amutant resulted in substantially decreased binding to ACTH. An emptycell indicates a mutant position that did not substantially alterbinding kinetics for that antibody. The rows corresponding to positions24 and beyond are not shown because none of these positions was observedto substantially alter antibody binding kinetics.

FIG. 42 shows the results of ¹²⁵I ACTH binding experiments demonstratingthat the tested anti-ACTH antibodies inhibited the binding of ACTH toMC2R expressing cells, as further described in Example 9. Each antibodytested is labeled on the X-axis and the level of binding detected isshown on the Y-axis.

FIG. 43 is a representative binding curve that shows neutralization ofACTH 1-24 induced signaling via MC2R (in this case, by Ab2).

FIG. 44 is a representative binding curve that shows neutralization ofACTH 1-24 induced signaling via MC2R (in this figure, by Ab13.H).

FIG. 45 provides representative binding data for the subject anti-humanACTH antibodies to ACTH 1-39 and the inability of human ACTH 1-13 andACTH 18-39 to compete with binding of ACTH 1-39 (specifically, forAb13).

FIG. 46 provides representative data showing the inhibition of ACTHdriven cAMP production in MC2R expressing cells (in this figure, byAb13.)

FIG. 47 provides representative data showing the inhibition of ACTHdriven cAMP production in MC1R expressing cells (in this figure, byAb13.)

FIG. 48 provides representative data showing the inhibition of ACTHdriven cAMP production in MC3R expressing cells (in this figure, byAb13.)

FIG. 49 provides representative data showing the inhibition of ACTHdriven cAMP production in MC4R expressing cells (in this figure, byAb13.)

FIG. 50 provides representative data showing the inhibition of ACTHdriven cAMP production in MC5R expressing cells (in this figure, byAb13.)

FIG. 51 provides representative data showing that the subject anti-ACTHantibodies (in this figure, Ab13.H) inhibited ACTH-induced cortisolproduction by Y1 cells.

FIG. 52 shows that Ab1.H inhibited ACTH-induced weight loss in the studydescribed in Example 13.

FIG. 53 shows plasma corticosterone levels before ACTH and antibodydosing in the study described in Example 13.

FIG. 54 shows plasma corticosterone levels at 24 hours after initiationof ACTH dosing and before the antibody administration in the studydescribed in Example 13.

FIG. 55 shows plasma corticosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration in the studydescribed in Example 13.

FIG. 56 shows plasma corticosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration in the studydescribed in Example 13.

FIG. 57 shows plasma corticosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration in the studydescribed in Example 13.

FIG. 58 shows plasma corticosterone levels 168 hours after initiation ofACTH dosing and 144 hours after the antibody administration in the studydescribed in Example 13.

FIG. 59 shows plasma aldosterone levels before ACTH and antibody dosingin the study described in Example 13.

FIG. 60 shows plasma aldosterone levels at 24 hours after initiation ofACTH dosing and before the antibody administration in the studydescribed in Example 13.

FIG. 61 shows plasma aldosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration in the studydescribed in Example 13.

FIG. 62 shows plasma aldosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration in the studydescribed in Example 13.

FIG. 63 shows plasma aldosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration in the studydescribed in Example 13.

FIG. 64 shows plasma aldosterone levels 168 hours after initiation ofACTH dosing and 144 hours after the antibody administration in the studydescribed in Example 13.

FIG. 65 shows the percentage change in animal weight by day, and showsthat Ab2.H, Ab11.H, and Ab12.H inhibited ACTH-induced weight loss forthe study described in Example 14.

FIG. 66 shows plasma corticosterone levels before ACTH and antibodydosing for animals treated with Ab2.H, Ab11.H, and Ab12.H as describedin Example 14.

FIG. 67 shows plasma corticosterone levels at 24 hours after initiationof ACTH dosing and before the antibody administration for animalstreated with Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 68 shows plasma corticosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 69 shows plasma corticosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 70 shows plasma corticosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration for animalstreated with Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 71 shows plasma aldosterone levels before ACTH and antibody dosingfor animals treated with Ab2.H, Ab11.H, and Ab12.H as described inExample 14.

FIG. 72 shows plasma aldosterone levels at 24 hours after initiation ofACTH dosing and before the antibody administration for animals treatedwith Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 73 shows plasma aldosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 74 shows plasma aldosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 75 shows plasma aldosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration for animalstreated with Ab2.H, Ab11.H, and Ab12.H as described in Example 14.

FIG. 76 shows the percentage change in animal weight by day, and showsthat Ab10.H inhibited ACTH-induced weight loss in the study described inExample 14.

FIG. 77 shows plasma corticosterone levels before ACTH and antibodydosing for animals treated with Ab10.H as described in Example 14.

FIG. 78 shows plasma corticosterone levels at 24 hours after initiationof ACTH dosing and before the antibody administration for animalstreated with Ab10.H as described in Example 14.

FIG. 79 shows plasma corticosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab10.H as described in Example 14.

FIG. 80 shows plasma corticosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab10.H as described in Example 14.

FIG. 81 shows plasma corticosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration for animalstreated with Ab10.H as described in Example 14.

FIG. 82 shows plasma aldosterone levels before ACTH and antibody dosingfor animals treated with Ab10.H as described in Example 14.

FIG. 83 shows plasma aldosterone levels at 24 hours after initiation ofACTH dosing and before the antibody administration for animals treatedwith Ab10.H as described in Example 14.

FIG. 84 shows plasma aldosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab10.H as described in Example 14.

FIG. 85 shows plasma aldosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab10.H as described in Example 14.

FIG. 86 shows plasma aldosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration for animalstreated with Ab10.H as described in Example 14.

FIG. 87 shows the percentage change in animal weight by day, and showsthat Ab13.H inhibited ACTH-induced weight loss for the study describedin Example 14.

FIG. 88 shows plasma corticosterone levels before ACTH and antibodydosing for animals treated with Ab13.H as described in Example 14.

FIG. 89 shows plasma corticosterone levels at 24 hours after initiationof ACTH dosing and before the antibody administration for animalstreated with Ab13.H as described in Example 14.

FIG. 90 shows plasma corticosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab13.H as described in Example 14.

FIG. 91 shows plasma corticosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab13.H as described in Example 14.

FIG. 92 shows plasma corticosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration for animalstreated with Ab13.H as described in Example 14.

FIG. 93 shows plasma aldosterone levels before ACTH and antibody dosingfor animals treated with Ab13.H as described in Example 14.

FIG. 94 shows plasma aldosterone levels at 24 hours after initiation ofACTH dosing and before the antibody administration for animals treatedwith Ab13.H as described in Example 14.

FIG. 95 shows plasma aldosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab13.H as described in Example 14.

FIG. 96 shows plasma aldosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab13.H as described in Example 14.

FIG. 97 shows plasma aldosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration for animalstreated with Ab13.H as described in Example 14.

FIG. 98 shows the percentage change in animal weight by day, and showsthat Ab7A.H inhibited ACTH-induced weight loss for the study describedin Example 14.

FIG. 99 shows plasma corticosterone levels before ACTH and antibodydosing for animals treated with Ab7A.H as described in Example 14.

FIG. 100 shows plasma corticosterone levels at 24 hours after initiationof ACTH dosing and before the antibody administration for animalstreated with Ab7A.H as described in Example 14.

FIG. 101 shows plasma corticosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab7A.H as described in Example 14.

FIG. 102 shows plasma corticosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab7A.H as described in Example 14.

FIG. 103 shows plasma corticosterone levels 144 hours after initiationof ACTH dosing and 120 hours after the antibody administration foranimals treated with Ab7A.H as described in Example 14.

FIG. 104 shows plasma aldosterone levels before ACTH and antibody dosingfor animals treated with Ab7A.H as described in Example 14.

FIG. 105 shows plasma aldosterone levels at 24 hours after initiation ofACTH dosing and before the antibody administration for animals treatedwith Ab7A.H as described in Example 14.

FIG. 106 shows plasma aldosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab7A.H as described in Example 14.

FIG. 107 shows plasma aldosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab7A.H as described in Example 14.

FIG. 108 shows plasma aldosterone levels 144 hours after initiation ofACTH dosing and 120 hours after the antibody administration for animalstreated with Ab7A.H as described in Example 14.

FIG. 109 shows plasma corticosterone levels before ACTH and antibodydosing for animals treated with Ab11A.H and Ab15.H as described inExample 14.

FIG. 110 shows plasma corticosterone levels at 24 hours after initiationof ACTH dosing and before the antibody administration for animalstreated with Ab11A.H and Ab15.H as described in Example 14.

FIG. 111 shows plasma corticosterone levels 48 hours after initiation ofACTH dosing and 24 hours after the antibody administration for animalstreated with Ab11A.H and Ab15.H as described in Example 14.

FIG. 112 shows plasma corticosterone levels 96 hours after initiation ofACTH dosing and 72 hours after the antibody administration for animalstreated with Ab11A.H and Ab15.H as described in Example 14.

FIG. 113 shows total Ab13.H antibody levels determined in each rat inthe pharmacokinetic study described in Example 15.

FIG. 114 shows the change in cortisol levels from baseline followinginjection of AD26-10 or Ab13.H over time in cynomolgus monkeys. Theresults demonstrate that Ab13.H reduced cortisol levels.

FIG. 115 shows the change in aldosterone levels from baseline followinginjection of AD26-10 or Ab13.H over time in cynomolgus monkeys. Theresults demonstrate that Ab13.H reduced aldosterone levels.

FIG. 116 shows the corticosterone levels observed over time in ratsdosed with AD26-10 or Ab13.H.

FIG. 117 shows the total antibody levels in rats over time followingadministration of AD26-10 or Ab13.H.

FIG. 118 shows plasma corticosterone levels in rabbits treated withvehicle (squares) or Ab13.H (circles). Corticosterone levels werereduced in the Ab13.H treatment group on days 10 and 11. Corticosteronelevels were significantly lower in the Ab13.H treatment group on day 15(p=0.0079) compared to control animals.

FIG. 119 shows the plasma corticosterone levels in rats treated withAD26-10 (square symbols) or Ab13.H (round symbols) and subjected to lowor high stress conditions. Plasma corticosterone levels weresignificantly reduced on days 2, 3, and 5 (all p=0.0002), and day 6(p=0.0068) in the Ab13.H treatment group compared to controls.

DETAILED DESCRIPTION

Antibodies and binding fragments thereof that bind to ACTH are disclosedherein. The antibody or antibody fragment according to the inventionbind to ACTH and prevent ACTH from functioning in various ways. In someembodiments, the antibody or antibody fragment neutralizes ACTH-inducedMCR signaling, inhibits ACTH-induced cortisol, corticosterone, and/oraldosterone secretion and/or reduces plasma cortisol, corticosterone,and/or aldosterone levels.

For convenience, the following sections generally outline the variousmeanings of the terms used herein. Following this discussion, generalaspects regarding antibodies or antibody fragments according to theinvention are discussed, followed by specific examples demonstrating theproperties of various embodiments of the antibodies or antibodyfragments according to the invention and how they can be employed.

Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

The terms “adrenocorticotropic hormone” or “adrenocorticotropin” or“adrenocorticotrophin” or “ACTH” or “ACTH 1-39” or “ACTH₁₋₃₉” or“corticotropin” or “corticotrophin” are used interchangeably and referto the polypeptide as set forth in SEQ ID NO:1121 as well as relatedpolypeptides, which include, but are not limited to, derivativevariants, substitution variants, deletion variants, and/or insertionvariants including the addition of an N-terminal methionine, fusionpolypeptides, and interspecies homologs. The terms “humanadrenocorticotropic hormone” or “human adrenocorticotropin” or “humanadrenocorticotrophin” or “hACTH” or “hACTH 1-39” or “hACTH₁₋₃₉” or“huACTH” or “huACTH 1-39” or “huACTH₁₋₃₉” are used interchangeably andrefer specifically to a human ACTH polypeptide such as the polypeptideas set forth in SEQ ID NO:1121. In certain embodiments, an ACTHpolypeptide includes terminal residues, such as, but not limited to,leader sequence residues, targeting residues, amino terminal methionineresidues, lysine residues, tag residues, and/or fusion protein residues.ACTH has also been referred to as corticotrophin or corticotropin. ACTHis a peptide hormone produced by post-translational enzymatic processingof POMC. In some tissues, e.g., the intermediate lobe, ACTH is furtherenzymatically processed to generate alpha-MSH and CLIP. Alpha-MSH hasthe same primary amino acid sequence as ACTH₁₋₁₃; however, two of theamino acids are modified in alpha-MSH, i.e., the N-terminal serine isacetylated and the C-terminal valine is amidated, but not ACTH₁₋₁₃. CLIPcorresponds to ACTH₁₈₋₃₉.

Except where the context indicates otherwise, the term “ACTH” as usedherein denotes the full-length human ACTH peptide containing 39 aminoacids (SYSMEHFRWGKPVGKKRRPVKVYPNGAEDESAEAFPLEF, SEQ ID NO:1121). ACTH isdistinct from “ACTH 1-13” (SYSMEHFRWGKPV, SEQ ID NO:1123), “ACTH 18-39”(RPVKVYPNGAEDESAEAFPLEF, SEQ ID NO: 1124) and “ACTH 1-24”(SYSMEHFRWGKPVGKKRRPVKVYP, SEQ ID NO:1122). However, the term alsorefers to the ACTH of another species when indicated by context, e.g.,equine ACTH or horse ACTH (Equus przewalskii, NCBI Accession No.XP_008513480), feline ACTH or cat ACTH (Felis catus, NCBI Accession No.XP_003984482), and canine ACTH or dog ACTH (Canus lupus familiaris, NCBIaccession no. AAK08973). The term ACTH also encompasses ACTH moleculesincorporating post-translational modifications, e.g., phosphorylation,glycosylation, ubiquitination, acetylation, methylation and/oramidation.

The term “human alpha-MSH” refers to a peptide that consists of aminoacids 1-13 of human ACTH. As discussed herein, alpha-MSH has the sameprimary amino acid sequence as amino acids 1-13 of human ACTH (alsoreferred to as “ACTH 1-13” or “ACTH₁₋₁₃”), but two of the amino acidsare modified in alpha-MSH, specifically, the N-terminal serine isacetylated and the C-terminal valine is amidated (having the sequenceSYSMEHFRWGKPV where S1 is acetylated and V13 is amidated, SEQ ID NO:1125). Except where context dictates otherwise, the terms “alpha-MSH”herein indicate human alpha-MSH.

The terms “human CLIP” or “human Corticotrophin-Like IntermediatePeptide” or “hACTH₁₈₋₃₉” or “hCLIP” or “ACTH 18-39” are usedinterchangeably and each refers to a peptide that consists of the 22C-terminal amino acid residues of human ACTH, i.e., amino acids 18-39 ofthe human ACTH polypeptide of SEQ ID NO:1121 (having the sequenceRPVKVYPNGAEDESAEAFPLEF, SEQ ID NO:1124). Except where context dictatesotherwise, the terms “CLIP” or “Corticotrophin-Like IntermediatePeptide” herein indicate human CLIP.

The term “anti-ACTH antibody or antibody fragment that does notsubstantially interact with or bind to at least one of ACTH₁₋₁₃,alpha-MSH, and/or ACTH₁₈₋₃₉ (CLIP)” means that the anti-ACTH antibody orantibody fragment binds to ACTH, typically human ACTH, with a bindingaffinity (K_(D)) that is substantially stronger than the bindingaffinity for said anti-ACTH antibody or antibody fragment to at leastone of ACTH₁₋₁₃, alpha-MSH, and/or ACTH₁₈₋₃₉ (CLIP), i.e., at least10-fold, 100-fold, 1000-fold or 10,000-fold stronger binding. Bindingaffinity may be expressed as “K_(D)” in molar units (e.g., nM or pM),with numerically lower values indicating stronger binding. Thus, a“stronger” affinity refers to a numerically lower K_(D) value, while a“weaker” affinity refers to a numerically higher K_(D) value. Inexemplary embodiments, said the binding affinity of said antibody forhuman ACTH will be at least 100-fold stronger than its binding affinityfor human CLIP and human alpha-MSH.

In some instances, this includes anti-ACTH antibodies or antibodyfragments thereof that do not detectably bind to ACTH₁₋₁₃, alpha-MSH,and/or ACTH₁₈₋₃₉ (CLIP) (e.g., several antibodies are designated ashaving a K_(D) of 1×10⁻¹ for CLIP in Table 5 or are designated as havinga K_(D) of 1×10⁻¹ for alpha-MSH in Table 6, which indicates nodetectable binding).

The term “cortisol” refers to a steroid hormone, more specifically aglucocorticoid, which is produced by the zona fasciculata of the adrenalcortex released in response to stress and a low level of blood glucose.The systematic (IUPAC) name of cortisol is(11β)-11,17,21-trihydroxypregn-4-ene-3,20-dione and its structure iswell known in the art and is shown below:

The term “Corticosterone” refers to a 21-carbon steroid hormone of thecorticosteroid type produced in the cortex of the adrenal glands inrodents and other non-human animals. The systematic (IUPAC) name ofcorticosterone is (11β)-11,21-dihydroxypregn-4-ene-3,20-dione and itsstructure is well known in the art and is shown below:

The term “aldosterone” refers is a steroid hormone of themineralocorticoid family which is produced by the outer section (zonaglomerulosa) of the adrenal cortex in the adrenal gland which plays arole in the regulation of blood pressure. The systematic (IUPAC) name ofaldosterone is 11β,21-Dihydroxy-3,20-dioxopregn-4-en-18-al and itsstructure is well known in the art and is shown below:

The terms “biological effects associated with ACTH” and “ACTH activity”are used interchangeably and include any biological effect of ACTH. Incertain embodiments, ACTH activity includes the ability of ACTH tointeract or bind to a receptor. In some embodiments, ACTH activity isrepresented by the ability of ACTH to bind to a melanocortin receptor(MCR). In some embodiments, ACTH binds to and activates MC2R in theadrenal cortex, thereby resulting in the production of cAMP, whichactivates PKA which in turn activates enzymes that convert cholesterolto cortisol, i.e., ACTH signaling through MC2R induces cortisolsecretion. ACTH can also bind to MC1R, MC3R, MC4R and/or MC5R and induceother biological effects.

The term “condition associated with elevated ACTH levels” refers to anycondition, disorder and disease present in a subject who also haselevated plasma ACTH levels. Elevated ACTH levels are often associatedwith elevated cortisol levels since ACTH is the primary stimulator ofadrenal cortisol production. ACTH and cortisol levels exhibit peaks (6-8a.m.) and nadirs (11 p.m.). Only a small percentage of circulatingcortisol is biologically active (i.e., free form), with the majority ofcortisol inactive (i.e., protein bound). Cortisol is inactivated in theliver and excreted in the urine as conjugated compounds (e.g.,17-hydroxysteroids). Urine free cortisol levels reflect circulating freeplasma cortisol levels. Since blood tests alone may not detect thepresence of excessive cortisol secretion (since levels naturally varythroughout the day), testing for elevated cortisol generally involves a24-hour urine free cortisol (UFC) measurement, cortisol saliva testingand blood tests. Measurement of ACTH levels, however, is most commonlyachieved by blood testing. Typically, blood will be drawn in the morningto obtain a peak ACTH level and/or drawn in the evening to obtain a low(trough) ACTH level. Normal values for ACTH blood levels range from 9-52pg/mL or 10-60 pg/mL for morning blood draws (there is no establishedreference value for evening blood draws). Higher than normal levels ofACTH may be present with hypertension, obstructive sleep apnea (OSA),congenital adrenal hyperplasia (CAH), Classical CAH, Nonclassical CAH,familial glucocorticoid deficiency (FGD), Allgrove syndrome, Nelson'sSyndrome, subsequent to bilateral adenectomy, Cushing's Disease, orCushing's Syndrome, and other diseases, disorders, and conditions.

As used herein, a “condition associated with ACTH” includes any disease,disorder, or condition that may be treated by antagonizing ACTH, forexample by administration of an anti-ACTH antibody or antigen-bindingfragment thereof according to the invention. Said disease, disorder, orcondition may be characterized by elevated ACTH. Said disease, disorder,or condition may be characterized by changes in the level of a substanceor in a biological process that can be ameliorated or reversed byantagonizing ACTH, including diseases, disorders, or conditionsassociated with elevated cortisol or aldosterone, wherein antagonism ofACTH may reduce said level of cortisol or aldosterone. Said diseases,disorders, or conditions include those associated with a symptom thatcan be ameliorated by antagonizing ACTH, whether or not ACTH is thoughtto play a causative role in the disease. Additional terms that are usedinterchangeably with “condition associated with ACTH” include “diseaseassociated with ACTH” as well as the terms “ACTH-related”,“ACTH-induced”, “ACTH-driven”, “ACTH-mediated” and “ACTH-associated”when used in the context of diseases, disorders, or conditions. Examplesof conditions associated with ACTH include, without limitation thereto,ACTH-driven hypercortisolism, acute coronary syndrome, acute heartfailure, Alzheimer's disease, anxiety disorders, atherosclerosis, atrialfibrillation, cachexia, cancer (such as Cushing's Syndrome resultingfrom ectopic ACTH expression, e.g., in small cell lung cancer, non-smallcell lung cancer (NSCLC), pancreatic carcinoma, neural tumors, orthymoma), cardiac conditions, cardiac fibrosis, cardiovasculardisorders, chronic renal failure, chronic stress syndrome, cognitivedysfunction, congenital adrenal hyperplasia (CAH), Classical CAH,Nonclassical CAH, familial glucocorticoid deficiency (FGD), Allgrovesyndrome, Nelson's syndrome, congestive heart failure, Conn's syndrome,coronary heart diseases, Cushing's Disease, Cushing's Syndrome,depression, diabetes, endothelial dysfunction, exercise intolerance,familial hyperaldosteronism, fibrosis, galactorrhea, heart failure,hyperaldosteronism, hypercortisolemia, hypertension, hypokalemia,impaired cardiac function, increased formation of collagen,inflammation, metabolic syndrome, muscle atrophy, conditions associatedwith muscle atrophy, myocardiac fibrosis, nephropathy, obesity,post-myocardial infarction, primary hyperaldosteronism, remodelingfollowing hypertension, renal failure, restenosis, secondaryhyperaldosteronism, sleep apnea, and syndrome X. Said conditionassociated with ACTH may be treated in a human, or in a non-human animalsuch as dog, cat, or horse, or another animal species.

The term “condition associated with elevated cortisol, corticosteroneand/or aldosterone levels” refers to any condition, disorder and diseasepresent in a subject who also has elevated plasma cortisol,corticosterone and/or aldosterone levels. Elevated aldosterone levels orhyperaldosteronism are associated with conditions such as primaryhyperaldosteronism (including Conn's syndrome), secondaryhyperaldosteronism, and familial hyperaldosteronism. Elevated cortisollevels, for example, are often associated with conditions such asanxiety disorders, stress, depression, obesity, cancer, muscle atrophy,hypertension, heart failures, diabetes, sleep apnea, hyperinsulinemia,Alzheimer's disease, dementia and other cognitive dysfunction,galactorrhea, metabolic syndrome, Cushing's Syndrome and Cushing'sDisease. Familial hyperaldosteronism includes a group of relatedheritable conditions that result in excessive production of aldosterone.Familial hyperaldosteronism patients often exhibit severe hypertension,and may exhibit enlarged adrenal glands. Familial hyperaldosteronism canbe categorized into three types, distinguished by their clinicalfeatures and genetic causes. In familial hyperaldosteronism type I,hypertension generally appears in childhood to early adulthood and canrange from mild to severe. This type can be treated with steroidmedications called glucocorticoids, so it is also known asglucocorticoid-remediable aldosteronism (GRA). One known genetic causeof familial hyperaldosteronism type I is the fusion the genes CYP11B1and CYP11B2, which are located close together on chromosome 8. Infamilial hyperaldosteronism type II, hypertension usually appears inearly to middle adulthood and does not improve with glucocorticoidtreatment. In most individuals with familial hyperaldosteronism typeIII, the adrenal glands are enlarged up to six times their normal size.These affected individuals have severe hypertension that starts inchildhood. The hypertension is difficult to treat and often results indamage to organs such as the heart and kidneys. Rarely, individuals withtype III have milder symptoms with treatable hypertension and no adrenalgland enlargement. Familial hyperaldosteronism type III can be caused bymutations in the KCNJ5 gene which encodes a potassium channel.

The term “Cushing's disease” refers to a serious condition of an excesslevel of the steroid hormone cortisol in the blood caused by a pituitarytumor secreting ACTH. Cushing's disease is rare, affecting 10 to 15people per million each year, most commonly adults between 20 and 50years of age. Women account for more than 70 percent of cases. Mostsubjects with Cushing's disease have small tumors (pituitarymicroadenomas). Cushing's disease is used exclusively to describe thecondition of excessive cortisol arising from a pituitary tumor secretingthe hormone ACTH. Magnetic resonance imaging (MRI) scan of the pituitarygland is the best way to detect the presence of an adenoma in Cushing'sdisease. MRI detects a pituitary adenoma in about 70 percent of cases.In the event that MRI scan fails to detect an abnormality despiteindications of Cushing's disease via clinical findings and hormonaltesting, inferior petrosal sinus sampling (IPSS) may be used to assessthe ACTH levels in the inferior petrosal sinus compared to a vein justbelow the heart. In Cushing's disease, the ACTH level in the inferiorpetrosal sinus is much higher compared to the vein below the heart.

Cushing's disease is not the same as Cushing's Syndrome. The term“Cushing's Syndrome” refers to the general state characterized byexcessive levels of cortisol in the blood. Elevated cortisol levels canoccur for reasons other than a pituitary tumor, including, e.g., tumorsof the adrenal glands producing cortisol; and ectopic ACTH production(i.e., certain types of cancer, elsewhere in the body, can make ACTH,which then stimulates the normal adrenal glands to make excessivecortisol). Cushing's Syndrome resulting from ectopic ACTH expression isfrequently cause by neoplasms including small cell lung cancer,non-small cell lung cancer (NSCLC), pancreatic carcinoma, neural tumors(such as gliomas, neuroepitheliomatous tumors, or nerve sheath tumors)and thymoma. Small cell lung cancer is a particularly prominent as ithas been observed to account for up to 50% of Cushing's Syndrome ofectopic or neoplastic origin.

Cushing's Syndrome is much more common than Cushing's disease. The mostcommon cause of elevated cortisol levels is taking medications that havecortisol, including, but not limited to, hydrocortisone, prednisonepills, skin ointments, asthma inhalers and joint steroid injections.Other, albeit less common, causes of elevated cortisol levels include,for example, an adrenal tumor or “Pseudo-Cushing's” (i.e., chronicallyelevated levels of cortisol due to, e.g., depression, alcohol abuse,anorexia nervosa or high estrogen levels).

The term “congenital adrenal hyperplasia” or CAH refers to an autosomalrecessive condition characterized by a congenital deficiency in cortisolproduction (reviewed in New et al. Congenital Adrenal Hyperplasia.[Updated 2013 Oct. 28]. In: De Groot et al., editors. Endotext[Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000. Availablefrom: www.ncbi.nlm.nih.gov/books/NBK278953/, last retrieved Aug. 19,2015, which is hereby incorporated by reference in its entirety).Genetic mutations within the biosynthesis pathway of cortisol have beenidentified as a cause of CAH. The most severe form of CAH is referred toas Classical CAH and is usually detected in the newborn or earlychildhood. Mutations in the cytochrome P450 21-hydroxylase gene (CYP21),which is important in the cortisol biosynthetic pathway, have beenreported as one common cause of Classical CAH. The milder form of CAH,called Non-classical CAH (NCAH), is due to a partial enzyme deficiencyand may cause symptoms at any time from infancy through adulthood. NCAHis much more common than Classical CAH. Cortisol provides negativefeedback for corticotropin-releasing hormone (CRH) and ACTH production.The deficiency in cortisol production in CAH patients leads to excessACTH secretion by the anterior pituitary as a result of this feedback,in an attempt to increase cortisol production. Thus, the lack ofcortisol in CAH patients can result in an elevation of ACTH levels. Thechronic high levels of ACTH can lead to an accumulation of cortisolprecursors, which in turn can result in increased androgen production.Specifically, the rise in ACTH stimulates the adrenal steroid pathway,but because of the defect in the cortisol biosynthesis pathway (e.g., ablock at 21-hydroxylation) there is a buildup in steroid precursors(such as 17-hydroxyprogesterone (17-OHP)), which can be androgenic.Thus, CAH patients typically exhibit low to no cortisol production,increased ACTH levels, and excess androgen production. This build up inandrogenic steroid precursors has important implications for the fetus,infant, child and adult with CAH.

For a female CAH patient, the buildup in androgens can result in avirilized fetus with ambiguous genitalia. In the infant and child theandrogens can cause pseudo-precocious puberty with excess growth andvirilization. Without effective treatment, the child will go through avery early puberty, resulting in a shortened stature. In the adult, CAHis associated with infertility, virilization of the female and steroiddeficiency.

Males with CAH, particularly if inadequately treated, may have reducedsperm counts and low testosterone as a result of small testes due tosuppression of gonadotropins and sometimes intra-testicular adrenalrests. All of these complications may result in diminished fertility(www.ncbi.nlm.nih.gov/books/NBK278953/). In addition high ACTH levels inCAH patients have been associated with testicular adrenal rest tumors(Delfino et. al., J. Ultrasound Med. 2012 March; 31(3):383-8.)

There are various treatment regimens available which attempt to provideadequate steroid levels during the day and counter the buildup of ACTHat night. A common regimen used in children is twice or thrice dailyhydrocortisone. This treatment regimen provides supra-physiologicallevels of hydrocortisone within 1-2 hours of dosing (Charmandari et al.,2001) and doesn't prevent the early morning rise in ACTH and androgenicprecursors (Scott et al., 1978; Cutler, 1996). Alternative treatmentregimens involve giving a dose of steroids at night, which does notreflect normal circadian rhythms and may affect the patient's sleepingpattern; additionally, such treatment regimens can incur a greater riskof giving excessive steroid doses, as the patient still receives steroidreplacement during the day.

Treatment of CAH involves steroid replacement, typically glucocorticoidreplacement. Steroid replacement can both functionally replace thelow/absent cortisol and reduce androgenic precursors. Glucocorticoidreplacement is typically administered to reduce hyperplasia and reduceoverproduction of androgens. The steroid may replacement therapy mayinclude hydrocortisone, prednisolone, or dexamethasone, or anothersteroid having glucocorticoid and/or mineralocorticoid activity. Steroidreplacement therapy at the dosages effective to treat CAH can causeside-effects, such as reducing growth in the child and causing thinbones and skin in the adult as well as potentially leading to Cushing'ssyndrome and/or metabolic syndrome. Moreover, even with cortisolsupplementation, ACTH levels may not be sufficiently suppressed tocontrol the excess androgen production.

Excess androgen production in CAH patients may be treated withantiandrogens (such as flutamide, gonadotropin-releasing hormoneanalogs, leuprolide, cyproterone (such as cyproterone acetate),enzalutamide, galeterone, abiraterone (e.g., abiraterone acetate),and/or orteronel); and/or non-steroidal antiandrogens (such asflutamide, VT-464, aminoglutethimide, and/or enzalutamide), and/oranother androgen receptor antagonist or androgen biosynthesis inhibitor.Patients may also be administered an aromatase inhibitor to slowskeletal maturation. Additionally, puberty may be suppressed byadministration of long-acting gonadotropin-releasing hormone (GnRH)agonists, and stimulating growth with growth hormone may partiallyimprove the patient's height. Replacement testosterone and/or estrogenmay be administered if the patient is deficient, e.g., at puberty.Additionally, salt wasting, if present, can be treated with dietarysupplementation with sodium chloride. Patients may alternatively, or inaddition, be administered an aldosterone replacement such asfludrocortisone (e.g., fludrocortisone acetate). Medication may beincreased in response to illness and/or stress (“stress-dosing”).

As further disclosed herein, the present disclosure provides a method oftreating CAH comprising administering an anti-ACTH antibody to a patientin need thereof. Without intent to be limited by theory, it is believedthat, by antagonizing ACTH in vivo, the anti-ACTH antibodies can preventthe effects of elevated ACTH present in CAH patients, includingstimulation of the adrenal steroid pathway, such as the buildup inandrogenic steroid precursors (e.g., 17-hydroxyprogesterone (17-OHP))can be decreased or prevented. Said anti-ACTH antibody may beadministered in an amount effective to treat CAH or to treat or preventone or more symptoms associated therewith, such as elevated productionof or levels of 17-hydroxyprogesterone (17-OHP) or another androgenicsteroid precursor, and/or elevated production of or levels of anandrogen such as testosterone, dihydrotestosterone (DHT) and/orandrostenedione, and/or virilization. Said patient may be furtheradministered one or more of: antiandrogens, flutamide,gonadotropin-releasing hormone analogs, leuprolide, cyproterone,cyproterone acetate, enzalutamide, galeterone, abiraterone, abirateroneacetate, orteronel, steroidal antiandrogens, flutamide, VT-464,aminoglutethimide, enzalutamide, an androgen receptor antagonist, anandrogen biosynthesis inhibitor, an aromatase inhibitor, a long-actinggonadotropin-releasing hormone (GnRH) agonists, and/or growth hormone,which optionally may be administered concurrently, sequentially,together or separately with said anti-ACTH antibody. Said patient may befurther administered replacement testosterone, dihydrotestosterone(DHT), androstenedione, and/or estrogen, which optionally may beadministered concurrently, sequentially, together or separately withsaid anti-ACTH antibody.

Familial glucocorticoid deficiency (FGD) or hereditary unresponsivenessto ACTH is characterized by isolated glucocorticoid deficiency andincludes FGF types 1 and 2. Rare, autosomal recessive forms of thisdisorder result from mutations in genes encoding either the ACTHreceptor (melanocortin 2 receptor (MC2R)) or its accessory protein(melanocortin 2 receptor accessory protein (MRAP)), which arerespectively categorized as FGD type 1 and 2. FGD patients do notrespond appropriately to ACTH and produce little to no cortisol. Sincecortisol negatively regulates ACTH, the lack of cortisol stimulates veryhigh levels of ACTH that has phenotypic effects (see Chung et al.,“Phenotypic characteristics of familial glucocorticoid deficiency (FGD)type 1 and 2.” Clinical Endocrinology, 72:589-594 (2010), which ishereby incorporated by reference in its entirety). FGD patientsfrequently present with hypoglycaemia, seizure, jaundice,hyperpigmentation, failure to thrive and frequent or severe infections.FGD patients also typically exhibit a markedly elevated plasma ACTH inthe presence of low cortisol but with a preserved mineralocorticoidproduction.

As further disclosed herein, the present disclosure provides a method oftreating FGD comprising administering an effective amount of ananti-ACTH antibody to a patient in need thereof. Without intent to belimited by theory, it is believed that, by antagonizing ACTH in vivo,the anti-ACTH antibodies can prevent the effects of elevated ACTHpresent in FGD patients, including but not limited to hypoglycaemia,seizure, jaundice, hyperpigmentation, failure to thrive and frequent orsevere infections. Said anti-ACTH antibody may be administered in anamount effective to treat FGD or to treat or prevent one or moresymptoms associated therewith. Optionally said treatment of FGD mayfurther comprise administration of glucocorticoid replacement therapy tothe patient, e.g., administration of one or more glucocorticoids and/oragents having glucocorticoid activity (such as agents having bothglucocorticoid and mineralocorticoid activity), such as cortisol(hydrocortisone), cortisone, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, and/or fludrocortisone (e.g., fludrocortisone acetate).

The present disclosure also provides a method of treating Nelson'ssyndrome (also known as post adrenalectomy syndrome) or elevated ACTHlevels subsequent to bilateral adenectomy, comprising administering aneffective amount of an anti-ACTH antibody to a patient in need thereof.Nelson's syndrome can occur in patients who have had both adrenal glandsremoved, e.g., for the treatment of Cushing's disease, sometimesoccurring many years after bilateral adrenalectomy. The disorder ischaracterized by elevated levels of ACTH. Without intent to be limitedby theory, it is believed that, by antagonizing ACTH in vivo, theanti-ACTH antibodies can prevent the effects of elevated ACTH present inNelson's syndrome patients. Said anti-ACTH antibody may be administeredin an amount effective to treat Nelson's syndrome or to treat or preventone or more symptoms associated therewith. Optionally said treatment mayfurther comprise administration of glucocorticoid replacement therapy tothe patient, e.g., one or more corticosteroids, includingglucocorticoids and/or mineralocorticoids (including agents having oneor both of glucocorticoid and/or mineralocorticoid activity), such ascortisol (hydrocortisone), cortisone, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone (e.g., fludrocortisone acetate),deoxycorticosterone (e.g., deoxycorticosterone acetate (DOCA)), and/oraldosterone.

The present disclosure also provides a method of treating triple Asyndrome, also known as Allgrove syndrome or “4 A” syndrome, comprisingadministering an effective amount of an anti-ACTH antibody to a patientin need thereof. This syndrome is characterized by the clinical triad ofadrenocorticotropic hormone (ACTH)-resistant adrenal failure, achalasiaof the cardia and alacrima, and further is associated with variable andprogressive neurological impairment involving the central, peripheraland autonomic nervous systems. The syndrome is also referred to in theliterature as “4 A” syndrome. Dermatological features such aspalmoplantar hyperkeratosis, as well as other signs including shortstature, osteoporosis and microcephaly, point to the multisystemiccharacter of the disorder which may severely impair life quality inaffected individuals. Allgrove syndrome patients exhibit chronicallyelevated levels of ACTH and, without intent to be limited by theory, itis believed that antagonism of ACTH, e.g., using an anti-ACTH antibody,may counteract some effects of the chronically elevated ACTH levels inAllgrove syndrome patients, thereby providing therapeutic benefit.

The term “sleep disorder” means any condition associated with irregularsleep patterns, e.g., sleep apnea, insomnia, hypersomnia, narcolepsy andother dyssomnias.

The term “sleep apnea” refers to a potentially serious sleep disorder inwhich breathing repeatedly stops and starts. There are two main types ofsleep apnea: (1) obstructive sleep apnea (OSA), which is the more commonform, that occurs when throat muscles relax; and (2) central sleep apnea(CSA), which occurs when your brain doesn't send proper signals to themuscles that control breathing. OSA occurs when the muscles in the backof the throat, which support the soft palate, the uvula, the tonsils,the side walls of the throat and the tongue, relax such that the airwaynarrows or closes preventing an adequate breath in. This may lower thelevel of oxygen in your blood. The brain senses the inability to breatheand briefly rouses a person from sleep in order to reopen the airway.The awakening is usually so brief that it is not remembered. In fact, aperson with OSA may not be aware that their sleep was disrupted, i.e.,some people with this type of sleep apnea think they sleep well allnight. A person may also make a snorting, choking or gasping sound. Thepattern of sleep/awake can repeat itself, e.g., 5 to 30 times or moreeach hour, all night. These disruptions impair the ability to reach thedesired deep, restful phases of sleep, and often result in a personsuffering from OSA feeling sleepy during their waking hours. CSA, whichis much less common than OSA, occurs when the brain fails to transmitsignals to the breathing muscles. A person with CSA may awaken withshortness of breath and/or have a difficult time getting to sleep orstaying asleep. As with OSA, snoring and daytime sleepiness can occur.The most common cause of CSA is heart failure and, less commonly, astroke. People with CSA may be more likely to remember awakening thanare people with OSA.

The signs and symptoms of OSA and CSA can overlap, which makes itdifficult to identify the type of sleep apnea. The most common signs andsymptoms of obstructive and central sleep apneas include: excessivedaytime sleepiness (hypersomnia); loud snoring (usually more prominentin OSA); episodes of breathing cessation during sleep witnessed byanother person; abrupt awakenings accompanied by shortness of breath(more likely indicates CSA); awakening with a dry mouth or sore throat;morning headache; difficulty staying asleep (insomnia); and/or attentionproblems.

Although sleep apnea can affect anyone, including children, there arecertain factors associated with an increased risk of sleep apnea. Riskfactors for OSA include, but are not limited to, excess weight (i.e.,fat deposits around your upper airway may obstruct your breathing); neckcircumference (i.e., people with a thicker neck may have a narrowerairway; a narrowed airway (i.e., a naturally narrow throat and/orenlarged tonsils or adenoids); gender (i.e., men are twice as likely aswoman to develop sleep apnea, although a woman's risk is increased ifshe is overweight and/or post-menopanusal); age (i.e., sleep apneaoccurs significantly more often in adults older than 60); family history(i.e., increased risk for individuals who have family members with sleepapnea); race (i.e., in people under 35 years old, people of Africandescent are more likely to have obstructive sleep apnea); use ofalcohol, sedatives or tranquilizers which relax the muscles in yourthroat; smoking (i.e., smokers are three times more likely to have OSAthan non-smokers due to, e.g., increased inflammation and fluidretention in the upper airway); nasal congestion (i.e., difficultybreathing through your nose, e.g., whether an anatomical problem orallergies, is associated with increased likelihood of developing OSA).Risk factors for CSA include, but are not limited to, gender (i.e.,males at increased risk); age (i.e., people over 65 years of age have ahigher risk of CSA); heart disorders (i.e., people with atrialfibrillation or congestive heart failure are more at risk of CSA); andstroke or brain tumor (i.e., these conditions can impair the brain'sability to regulate breathing.

Sleep apnea is considered a serious medical condition with complicationsincluding, but not limited to, high blood pressure (i.e., hypertension)and heart problems, daytime fatigue, depression, behavioral problems,problems with medications and/or surgery, liver problems andsleep-deprived partners.

“About” where used means especially ±10%, ±5% or ±3% (referring to thegiven numeric value, respectively), if not indicated otherwise. In eachof the invention embodiments, “about” can be deleted.

The term “host cell” herein in general refers to any cell engineered toexpress one or more antibody polypeptides according to the invention.This includes by way of example bacterial, fungal, yeast, mammalian,invertebrate such as insect, plant and avian cells. Preferred host cellsare yeast, fungi, especially filamentous fungi and mammalian cells.Yeast and filamentous fungi include, but are not limited to Pichiapastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae,Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichialindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria,Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica,Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenulapolymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans,Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichodermareesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum,Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichiasp., any Saccharomyces sp., Hansenula polymorpha, any Kluyveromyces sp.,Candida albicans, any Aspergillus sp., Trichoderma reesei, Chrysosporiumlucknowense, any Fusarium sp. and Neurospora crassa.

Examples of invertebrate cells include insect cells such as DrosophilaS2 and Spodoptera Sf9, as well as plant cells. Examples of usefulmammalian host cell lines include Chinese hamster ovary (CHO) and COScells. More specific examples include monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlauband Chasin, PNAS USA, 77:4216 (1980)); mouse Sertoli cells (TM4, Mather,Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75);human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art. Preferred mammalian cells forantibody expression include CHO cells and COS cells. In an exemplaryembodiment the recombinant host cells are polyploid yeast cells of thegenus Pichia.

Mating competent yeast species: In the present invention this isintended to broadly encompass any diploid or tetraploid yeast which canbe grown in culture. Such species of yeast may exist in a haploid,diploid, or other polyploid form. The cells of a given ploidy may, underappropriate conditions, proliferate for an indefinite number ofgenerations in that form. Diploid cells can also sporulate to formhaploid cells. Sequential mating can result in tetraploid strainsthrough further mating or fusion of diploid strains. The presentinvention contemplates the use of haploid yeast, as well as diploid orother polyploid yeast cells produced, for example, by mating orspheroplast fusion.

Mating competent yeast include yeast which are a member of theSaccharomycetaceae family, which includes the genera Arxiozyma;Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera; Eremothecium;Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces;Pachysolen; Pichia; Saccharomyces; Saturnispora; Tetrapisispora;Torulaspora; Williopsis; and Zygosaccharomyces. Other types of yeastpotentially useful in the invention include Yarrowia; Rhodosporidium;Candida; Hansenula; Filobasium; Sporidiobolus; Bullera; Leucosporidiumand Filobasidella.

In a preferred embodiment of the invention, the mating competent yeastis a member of the genus Pichia. In a further preferred embodiment ofthe invention, the mating competent yeast of the genus Pichia is one ofthe following species: Pichia pastoris, Pichia methanolica, andHansenula polymorpha (Pichia angusta). In a particularly preferredembodiment of the invention, the mating competent yeast of the genusPichia is the species Pichia pastoris.

Haploid Yeast Cell: A cell having a single copy of each gene of itsnormal genomic (chromosomal) complement.

Polyploid Yeast Cell: A cell having more than one copy of its normalgenomic (chromosomal) complement.

Diploid Yeast Cell: A cell having two copies (alleles) of essentiallyevery gene of its normal genomic complement, typically formed by theprocess of fusion (mating) of two haploid cells.

Tetraploid Yeast Cell: A cell having four copies (alleles) ofessentially every gene of its normal genomic complement, typicallyformed by the process of fusion (mating) of two haploid cells.Tetraploids may carry two, three, four or more different expressioncassettes. Such tetraploids might be obtained in S. cerevisiae byselective mating homozygotic heterothallic a/a and alpha/alpha diploidsand in Pichia by sequential mating of haploids to obtain auxotrophicdiploids. For example, a [met his] haploid can be mated with [ade his]haploid to obtain diploid [his]; and a [met arg] haploid can be matedwith [ade arg] haploid to obtain diploid [arg]; then the diploid[his]×diploid [arg] to obtain a tetraploid prototroph. It will beunderstood by those of skill in the art that reference to the benefitsand uses of diploid cells may also apply to tetraploid cells.

Yeast Mating: The process by which two haploid yeast cells naturallyfuse to form one diploid yeast cell.

Meiosis: The process by which a diploid yeast cell undergoes reductivedivision to form four haploid spore products. Each spore may thengerminate and form a haploid vegetatively growing cell line.

Selectable Marker: A selectable marker is a gene or gene fragment thatconfers a growth phenotype (physical growth characteristic) on a cellreceiving that gene as, for example through a transformation event. Theselectable marker allows that cell to survive and grow in a selectivegrowth medium under conditions in which cells that do not receive thatselectable marker gene cannot grow. Selectable marker genes generallyfall into several types, including positive selectable marker genes suchas a gene that confers on a cell resistance to an antibiotic or otherdrug, temperature when two temperature sensitive (“ts”) mutants arecrossed or a ts mutant is transformed; negative selectable marker genessuch as a biosynthetic gene that confers on a cell the ability to growin a medium without a specific nutrient needed by all cells that do nothave that biosynthetic gene, or a mutagenized biosynthetic gene thatconfers on a cell inability to grow by cells that do not have the wildtype gene; and the like. Suitable markers include but are not limitedto: ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3; URA3; and the like.

Expression Vector: These DNA vectors contain elements that facilitatemanipulation for the expression of a foreign protein within the targethost cell. Conveniently, manipulation of sequences and production of DNAfor transformation is first performed in a bacterial host, e.g. E. coli,and usually vectors will include sequences to facilitate suchmanipulations, including a bacterial origin of replication andappropriate bacterial selection marker. Selection markers encodeproteins necessary for the survival or growth of transformed host cellsgrown in a selective culture medium. Host cells not transformed with thevector containing the selection gene will not survive in the culturemedium. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media. Exemplary vectors and methods for transformation of yeastare described, for example, in Burke, D., Dawson, D., & Stearns, T.(2000). Methods in yeast genetics: a Cold Spring Harbor Laboratorycourse manual. Plainview, N.Y.: Cold Spring Harbor Laboratory Press.

Expression vectors for use in the methods of the invention will furtherinclude yeast specific sequences, including a selectable auxotrophic ordrug marker for identifying transformed yeast strains. A drug marker mayfurther be used to amplify copy number of the vector in a yeast hostcell.

The polypeptide coding sequence of interest is operably linked totranscriptional and translational regulatory sequences that provide forexpression of the polypeptide in yeast cells. These vector componentsmay include, but are not limited to, one or more of the following: anenhancer element, a promoter, and a transcription termination sequence.Sequences for the secretion of the polypeptide may also be included,e.g. a signal sequence, and the like. A yeast origin of replication isoptional, as expression vectors are often integrated into the yeastgenome. In one embodiment of the invention, the polypeptide of interestis operably linked, or fused, to sequences providing for optimizedsecretion of the polypeptide from yeast diploid cells.

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (Gateway Technology; Invitrogen, Carlsbad Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequences to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters(that increase levels of transcription in response to absence of arepressor). Inducible promoters may initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, e.g., the presence or absence of a nutrient or achange in temperature.

The promoter fragment may also serve as the site for homologousrecombination and integration of the expression vector into the samesite in the host genome; alternatively a selectable marker is used asthe site for homologous recombination.

Examples of suitable promoters useful in Pichia include the AOX1promoter (Cregg et al. (1989) Mol. Cell. Biol. 9:1316-1323); ICL1promoter (Menendez et al. (2003) Yeast 20(13):1097-108);glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham et al.(1997) Gene 186(1):37-44); and FLD1 promoter (Shen et al. (1998) Gene216(1):93-102). The GAP promoter is a strong constitutive promoter andthe AOX and FLD1 promoters are inducible.

Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4,PHO3, PHO5, Pyk, and chimeric promoters derived therefrom. Additionally,non-yeast promoters may be used in the invention such as mammalian,insect, plant, reptile, amphibian, bacterial, fungal, viral, and avianpromoters. Most typically the promoter will comprise a mammalianpromoter (potentially endogenous to the expressed genes) or willcomprise a yeast or viral promoter that provides for efficienttranscription in yeast systems.

The polypeptides of interest may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, e.g. a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the polypeptide coding sequence that isinserted into the vector. The heterologous signal sequence selectedpreferably is one that is recognized and processed through one of thestandard pathways available within the host cell. The S. cerevisiaealpha factor pre-pro signal has proven effective in the secretion of avariety of recombinant proteins from P. pastoris. Other yeast signalsequences include the alpha mating factor signal sequence, the invertasesignal sequence, and signal sequences derived from other secreted yeastpolypeptides. Additionally, these signal peptide sequences may beengineered to provide for enhanced secretion in diploid yeast expressionsystems. Other secretion signals of interest also include mammaliansignal sequences, which may be heterologous to the protein beingsecreted, or may be a native sequence for the protein being secreted.Signal sequences include pre-peptide sequences, and in some instancesmay include propeptide sequences. Many such signal sequences are knownin the art, including the signal sequences found on immunoglobulinchains, e.g., K28 preprotoxin sequence, PHA-E, FACE, human MCP-1, humanserum albumin signal sequences, human Ig heavy chain, human Ig lightchain, and the like. For example, see Hashimoto et. al., Protein Eng11(2) 75 (1998); and Kobayashi et. al., Therapeutic Apheresis 2(4) 257(1998).

Transcription may be increased by inserting a transcriptional activatorsequence into the vector. These activators are cis-acting elements ofDNA, usually about from 10 to 300 bp, which act on a promoter toincrease its transcription. Transcriptional enhancers are relativelyorientation and position independent, having been found 5′ and 3′ to thetranscription unit, within an intron, as well as within the codingsequence itself. The enhancer may be spliced into the expression vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from 3′ tothe translation termination codon, in untranslated regions of eukaryoticor viral DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques orPCR/recombination methods. Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required or via recombination methods. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures areused to transform host cells, and successful transformants selected byantibiotic resistance (e.g. ampicillin or Zeocin) where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionendonuclease digestion and/or sequenced.

As an alternative to restriction and ligation of fragments,recombination methods based on att sites and recombination enzymes maybe used to insert DNA sequences into a vector. Such methods aredescribed, for example, by Landy (1989) Ann. Rev. Biochem. 58:913-949;and are known to those of skill in the art. Such methods utilizeintermolecular DNA recombination that is mediated by a mixture of lambdaand E. coli-encoded recombination proteins. Recombination occurs betweenspecific attachment (att) sites on the interacting DNA molecules. For adescription of att sites see Weisberg and Landy (1983) Site-SpecificRecombination in Phage Lambda, in Lambda II, Weisberg, ed. (Cold SpringHarbor, N.Y.: Cold Spring Harbor Press), pp. 211-250. The DNA segmentsflanking the recombination sites are switched, such that afterrecombination, the att sites are hybrid sequences comprised of sequencesdonated by each parental vector. The recombination can occur betweenDNAs of any topology.

Att sites may be introduced into a sequence of interest by ligating thesequence of interest into an appropriate vector; generating a PCRproduct containing att B sites through the use of specific primers;generating a cDNA library cloned into an appropriate vector containingatt sites; and the like.

Folding, as used herein, refers to the three-dimensional structure ofpolypeptides and proteins, where interactions between amino acidresidues act to stabilize the structure. While non-covalent interactionsare important in determining structure, usually the proteins of interestwill have intra- and/or intermolecular covalent disulfide bonds formedby two cysteine residues. For naturally occurring proteins andpolypeptides or derivatives and variants thereof, the proper folding istypically the arrangement that results in optimal biological activity,and can conveniently be monitored by assays for activity, e.g. ligandbinding, enzymatic activity, etc.

In some instances, for example where the desired product is of syntheticorigin, assays based on biological activity will be less meaningful. Theproper folding of such molecules may be determined on the basis ofphysical properties, energetic considerations, modeling studies, and thelike.

The expression host may be further modified by the introduction ofsequences encoding one or more enzymes that enhance folding anddisulfide bond formation, i.e. foldases, chaperonins, etc. Suchsequences may be constitutively or inducibly expressed in the yeast hostcell, using vectors, markers, etc. as known in the art. Preferably thesequences, including transcriptional regulatory elements sufficient forthe desired pattern of expression, are stably integrated in the yeastgenome through a targeted methodology.

For example, the eukaryotic PDI is not only an efficient catalyst ofprotein cysteine oxidation and disulfide bond isomerization, but alsoexhibits chaperone activity. Co-expression of PDI can facilitate theproduction of active proteins having multiple disulfide bonds. Also ofinterest is the expression of BIP (immunoglobulin heavy chain bindingprotein); cyclophilin; and the like. In one embodiment of the invention,each of the haploid parental strains expresses a distinct foldingenzyme, e.g. one strain may express BIP, and the other strain mayexpress PDI or combinations thereof.

The terms “desired protein” or “desired antibody” are usedinterchangeably and refer generally to a parent antibody or fragmentspecific to a target, i.e., ACTH or a chimeric or humanized antibody ora binding portion thereof derived therefrom or one containing the sameCDRs or epitopic specificity as any of the anti-ACTH antibodies orfragments described herein. The term “antibody” is intended to includeany polypeptide chain-containing molecular structure with a specificshape that fits to and recognizes an epitope, where one or morenon-covalent binding interactions stabilize the complex between themolecular structure and the epitope. The archetypal antibody molecule isthe immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA,IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow,sheep, pig, dog, other mammals, chicken, other avians, etc., areconsidered to be “antibodies.” A preferred source for producingantibodies useful as starting material according to the invention israbbits. Numerous antibody coding sequences have been described; andothers may be raised by methods well-known in the art. Examples thereofinclude chimeric antibodies, human antibodies and other non-humanmammalian antibodies, humanized antibodies, single chain antibodies(such as scFvs), camelbodies, nanobodies, IgNAR (single-chain antibodiesderived from sharks), small-modular immunopharmaceuticals (SMIPs), andantibody fragments such as Fabs, Fab′, F(ab′)₂, monovalent antibodyfragments such as MetMab like molecules, and the like. See Streltsov VA, et al., Structure of a shark IgNAR antibody variable domain andmodeling of an early-developmental isotype, Protein Sci. 2005 November;14(11):2901-9. Epub 2005 Sep. 30; Greenberg A S, et al., A new antigenreceptor gene family that undergoes rearrangement and extensive somaticdiversification in sharks, Nature. 1995 Mar. 9; 374(6518): 168-73;Nuttall S D, et al., Isolation of the new antigen receptor fromwobbegong sharks, and use as a scaffold for the display of protein looplibraries, Mol Immunol. 2001 August; 38(4):313-26; Hamers-Casterman C,et al., Naturally occurring antibodies devoid of light chains, Nature.1993 Jun. 3; 363(6428):446-8; Gill D S, et al., Biopharmaceutical drugdiscovery using novel protein scaffolds, Curr Opin Biotechnol. 2006December; 17(6):653-8. Epub 2006 Oct. 19.

The present invention includes in particular includes monovalentantibody molecules that bind ACTH, which are analogous to MetMabmolecules. MetMab is a monovalent antibody specific to Met. (Met is aprotein encoded by the nucleotide sequence set forth in Park et al.,PNAS USA 84, 6379-83 (1987), or fragments thereof, as well as relatedpolypeptides, which include, but are not limited to, allelic variants,splice variants, derivative variants, substitution variants, deletionvariants, and/or insertion variants, fusion polypeptides, andinterspecies homologs). The MetMab antibody, is a monovalent antibodyknown by different names including OA-5d5 (Genentech) and is also calledOne Armed 5d5, 5d5, MetMab, PRO143966, among others). Antibody OA-5d5,including its structure and properties, and methods for making and usingit, are described in U.S. Publication No. 2007/0092520. In oneembodiment, an anti-ACTH antibody according to the invention maycomprise a single Fab region linked to an Fc region. In such embodiment,an antibody of the invention may comprise light and heavy chain variabledomains as described herein. In such an embodiment, the antibody ismonovalent and may comprise an intact Fc region. In another suchembodiment, the Fc region may comprise at least one protuberance (knob)and at least one cavity (hole), wherein the presence of the protuberanceand cavity enhances formation of a complex between an Fc polypeptidecomprising the protuberance and an Fc polypeptide comprising the cavity,for example as described in WO 2005/063816. In one embodiment, the Fcregion of an antibody of the invention may comprise a first and a secondFc polypeptide, wherein the first and second polypeptide each comprisesone or more mutations with respect to wild type human Fc. In oneembodiment, a cavity mutation is T366S, L368A and/or Y407V. In anotherembodiment, a protuberance mutation is T366W. In a specific embodiment,a monovalent antibody according to the subject invention may comprise aone-armed antibody synthesized as described in WO2005/063816. In suchembodiment, the one-armed antibody may comprise Fc mutationsconstituting “knobs” and “holes” as described in WO2005/063816. Forexample, a hole mutation can be one or more of T366A, L368A and/or Y407Vin an Fc polypeptide, and a cavity mutation can be T366W. The inventionis also directed to an anti-human ACTH monovalent agent that binds withthe same ACTH epitope and/or competes with an anti-ACTH antibody forbinding to ACTH as an antibody or antibody fragment disclosed herein.

For example, antibodies or antigen binding fragments may be produced bygenetic engineering. In this technique, as with other methods,antibody-producing cells are sensitized to the desired antigen orimmunogen. The messenger RNA isolated from antibody producing cells isused as a template to make cDNA using PCR amplification. A library ofvectors, each containing one heavy chain gene and one light chain generetaining the initial antigen specificity, is produced by insertion ofappropriate sections of the amplified immunoglobulin cDNA into theexpression vectors. A combinatorial library is constructed by combiningthe heavy chain gene library with the light chain gene library. Thisresults in a library of clones which co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost cell. When antibody gene synthesis is induced in the transfectedhost, the heavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

Antibody coding sequences of interest include those encoded by nativesequences, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(aa) substitutions, additions or deletions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions toeliminate non-essential amino acids, such as to alter a glycosylationsite, or to minimize misfolding by substitution or deletion of one ormore cysteine residues that are not necessary for function. Variants canbe designed so as to retain or have enhanced biological activity of aparticular region of the protein (e.g., a functional domain, catalyticamino acid residues, etc). Variants also include fragments of thepolypeptides disclosed herein, particularly biologically activefragments and/or fragments corresponding to functional domains.Techniques for in vitro mutagenesis of cloned genes are known. Alsoincluded in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.

As used herein, the terms “chimeric antibodies” and “chimerizedantibodies” (as well as the respective singular forms thereof) are usedinterchangeably and have the same meaning. Chimeric antibodies generallycomprise one or more variable domains of one species origin and aconstant domain of another species origin. Most typically a chimericantibody comprises variable heavy and variable light chain antibodies ofnon-human (e.g., rabbit, or rodent) one or both of which are linked to aconstant domain of another species origin (e.g., human). Exemplarychimeric antibodies comprise a variable heavy chain of rabbit originlinked (e.g., fused) to a constant heavy chain of human origin, and mayfurther contain a variable light chain of rabbit origin which may belinked (e.g., fused) to a light chain of human origin (or rabbitorigin).

Chimeric antibodies may be made by recombinant means by combining thevariable light and heavy chain regions (V_(L) and V_(H)), obtained fromantibody producing cells of one species with the constant light andheavy chain regions from another. Typically chimeric antibodies utilizerodent or rabbit variable regions and human constant regions, in orderto produce an antibody with predominantly human domains. The productionof such chimeric antibodies is well known in the art, and may beachieved by standard means (as described, e.g., in U.S. Pat. No.5,624,659, incorporated herein by reference in its entirety). It isfurther contemplated that the human constant regions of chimericantibodies of the invention may be selected from IgG1, IgG2, IgG3, andIgG4 constant regions.

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate primarily thecomplementarity-determining regions of the animal-derived antibody. Thisis accomplished by carefully examining the sequence of thehyper-variable loops of the variable regions of the monoclonal antibody,and grafting them to the human antibody frameworks that are most similarto the rabbit sequence present in the particular antibody. This can alsobe achieved by fitting the CDRs to the structure of the human antibodychains. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully herein byreference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, Fab, or other fragments) may be synthesized.“Fragment” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing a fusedvariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities. In another embodiment of theinvention, SMIPs (small molecule immunopharmaceuticals), camelbodies,nanobodies, and IgNAR are encompassed by immunoglobulin fragments.

Immunoglobulins and fragments thereof may be modifiedpost-translationally, e.g. to add effector moieties such as chemicallinkers, detectable moieties, such as fluorescent dyes, enzymes, toxins,substrates, bioluminescent materials, radioactive materials,chemiluminescent moieties and the like, or specific binding moieties,such as streptavidin, avidin, or biotin, and the like may be utilized inthe methods and compositions of the present invention. Examples ofadditional effector molecules are provided infra.

A polynucleotide sequence “corresponds” to a polypeptide sequence iftranslation of the polynucleotide sequence in accordance with thegenetic code yields the polypeptide sequence (i.e., the polynucleotidesequence “encodes” the polypeptide sequence), one polynucleotidesequence “corresponds” to another polynucleotide sequence if the twosequences encode the same polypeptide sequence.

A “heterologous” region or domain of a DNA construct is an identifiablesegment of DNA within a larger DNA molecule that is not found inassociation with the larger molecule in nature. Thus, when theheterologous region encodes a mammalian gene, the gene will usually beflanked by DNA that does not flank the mammalian genomic DNA in thegenome of the source organism. Another example of a heterologous regionis a construct where the coding sequence itself is not found in nature(e.g., a cDNA where the genomic coding sequence contains introns, orsynthetic sequences having codons different than the native gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

A “coding sequence” is an in-frame sequence of codons that (in view ofthe genetic code) correspond to or encode a protein or peptide sequence.Two coding sequences correspond to each other if the sequences or theircomplementary sequences encode the same amino acid sequences. A codingsequence in association with appropriate regulatory sequences may betranscribed and translated into a polypeptide. A polyadenylation signaland transcription termination sequence will usually be located 3′ to thecoding sequence. A “promoter sequence” is a DNA regulatory regioncapable of binding RNA polymerase in a cell and initiating transcriptionof a downstream (3′ direction) coding sequence. Promoter sequencestypically contain additional sites for binding of regulatory molecules(e.g., transcription factors) which affect the transcription of thecoding sequence. A coding sequence is “under the control” of thepromoter sequence or “operatively linked” to the promoter when RNApolymerase binds the promoter sequence in a cell and transcribes thecoding sequence into mRNA, which is then in turn translated into theprotein encoded by the coding sequence.

Vectors are used to introduce a foreign substance, such as DNA, RNA orprotein, into an organism or host cell. Typical vectors includerecombinant viruses (for polynucleotides) and liposomes (forpolypeptides). A “DNA vector” is a replicon, such as plasmid, phage orcosmid, to which another polynucleotide segment may be attached so as tobring about the replication of the attached segment. An “expressionvector” is a DNA vector which contains regulatory sequences which willdirect polypeptide synthesis by an appropriate host cell. This usuallymeans a promoter to bind RNA polymerase and initiate transcription ofmRNA, as well as ribosome binding sites and initiation signals to directtranslation of the mRNA into a polypeptide(s). Incorporation of apolynucleotide sequence into an expression vector at the proper site andin correct reading frame, followed by transformation of an appropriatehost cell by the vector, enables the production of a polypeptide encodedby said polynucleotide sequence.

“Amplification” of polynucleotide sequences is the in vitro productionof multiple copies of a particular nucleic acid sequence. The amplifiedsequence is usually in the form of DNA. A variety of techniques forcarrying out such amplification are described in a review article by VanBrunt (1990, Bio/Technol., 8(4):291-294). Polymerase chain reaction orPCR is a prototype of nucleic acid amplification, and use of PCR hereinshould be considered exemplary of other suitable amplificationtechniques.

The general structure of antibodies in vertebrates now is wellunderstood (Edelman, G. M., Ann. N.Y. Acad. Sci., 190: 5 (1971)).Antibodies consist of two identical light polypeptide chains ofmolecular weight approximately 25,000 Daltons (the “light chain”), andtwo identical heavy chains of molecular weight approximately 50,000Daltons (the “heavy chain”). The four chains are joined by disulfidebonds in a “Y” configuration wherein the light chains bracket the heavychains starting at the mouth of the “Y” configuration. The “branch”portion of the “Y” configuration is designated the F_(ab) region; thestem portion of the “Y” configuration is designated the Fc region. Theamino acid sequence orientation runs from the N-terminal end at the topof the “Y” configuration to the C-terminal end at the bottom of eachchain. The N-terminal end possesses the variable region havingspecificity for the antigen that elicited it, and is approximately 100amino acids in length, there being slight variations between light andheavy chain and from antibody to antibody.

The variable region is linked in each chain to a constant region thatextends the remaining length of the chain and that within a particularclass of antibody does not vary with the specificity of the antibody(i.e., the antigen eliciting it). There are five known major classes ofconstant regions that determine the class of the immunoglobulin molecule(IgG, IgM, IgA, IgD, and IgE corresponding to γ, μ, α, δ, and ε (gamma,mu, alpha, delta, or epsilon) heavy chain constant regions). Theconstant region or class determines subsequent effector function of theantibody, including activation of complement (Kabat, E. A., StructuralConcepts in Immunology and Immunochemistry, 2nd Ed., p. 413-436, Holt,Rinehart, Winston (1976)), and other cellular responses (Andrews, D. W.,et al., Clinical Immunobiology, pp 1-18, W. B. Sanders (1980); Kohl, S.,et al., Immunology, 48: 187 (1983)); while the variable regiondetermines the antigen with which it will react. Light chains areclassified as either κ (kappa) or λ (lambda). Each heavy chain class canbe prepared with either kappa or lambda light chain. The light and heavychains are covalently bonded to each other, and the “tail” portions ofthe two heavy chains are bonded to each other by covalent disulfidelinkages when the immunoglobulins are generated either by hybridomas orby B cells.

The expression “variable region” or “VR” refers to the domains withineach pair of light and heavy chains in an antibody that are involveddirectly in binding the antibody to the antigen. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain (V_(L)) at one end and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain.

The expressions “complementarity determining region,” “hypervariableregion,” or “CDR” refer to one or more of the hyper-variable orcomplementarity determining regions (CDRs) found in the variable regionsof light or heavy chains of an antibody (See Kabat, E. A. et al.,Sequences of Proteins of Immunological Interest, National Institutes ofHealth, Bethesda, Md., (1987)). These expressions include thehypervariable regions as defined by Kabat et al. (“Sequences of Proteinsof Immunological Interest,” Kabat E., et al., US Dept. of Health andHuman Services, 1983) or the hypervariable loops in 3-dimensionalstructures of antibodies (Chothia and Lesk, J. Mol. Biol. 196 901-917(1987)). The CDRs in each chain are held in close proximity by frameworkregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site. Within the CDRs there are selectamino acids that have been described as the selectivity determiningregions (SDRs) which represent the critical contact residues used by theCDR in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34(2005)).

An “epitope” or “binding site” is an area or region on an antigen towhich an antigen-binding peptide (such as an antibody) specificallybinds. A protein epitope may comprise amino acid residues directlyinvolved in the binding (also called immunodominant component of theepitope) and other amino acid residues, which are not directly involvedin the binding, such as amino acid residues which are effectivelyblocked by the specifically antigen binding peptide (in other words, theamino acid residue is within the “footprint” of the specifically antigenbinding peptide). The term epitope herein includes both types of aminoacid binding sites in any particular region of ACTH that specificallybinds to an anti-ACTH antibody. ACTH may comprise a number of differentepitopes, which may include, without limitation, (1) linear peptideantigenic determinants, (2) conformational antigenic determinants whichconsist of one or more non-contiguous amino acids located near eachother in a mature ACTH conformation; and (3) post-translationalantigenic determinants which consist, either in whole or part, ofmolecular structures covalently attached to an ACTH protein such ascarbohydrate groups.

The phrase that a first antibody binds “substantially” or “at leastpartially” the same epitope as a second antibody means that the epitopebinding site for the first antibody comprises at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or more of the amino acid residues on theantigen that constitutes the epitope binding site of the secondantibody. Also, that a first antibody binds substantially or partiallythe same or overlapping epitope as a second antibody means that thefirst and second antibodies compete in binding to the antigen, asdescribed above. Thus, the term “binds to substantially the same epitopeor determinant as” a monoclonal antibody means that an antibody“competes” with the antibody.

The phrase “binds to the same or overlapping epitope or determinant as”an antibody of interest means that an antibody “competes” with saidantibody of interest for at least one, (e.g., at least 2, at least 3, atleast 4, at least 5) or all residues on ACTH to which said antibody ofinterest specifically binds. The identification of one or moreantibodies that bind(s) to substantially or essentially the same epitopeas the monoclonal antibodies described herein can be readily determinedusing alanine scanning. Additionally, any one of variety ofimmunological screening assays in which antibody competition can beassessed. A number of such assays are routinely practiced and well knownin the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997,which is specifically incorporated herein by reference). It will beunderstood that actually determining the epitope to which an antibodydescribed herein binds is not in any way required to identify anantibody that binds to the same or substantially the same or overlappingepitope as the monoclonal antibody described herein.

For example, where the test antibodies to be examined are obtained fromdifferent source animals, or are even of a different Ig isotype, asimple competition assay may be employed in which the control antibodyis mixed with the test antibody and then applied to a sample containingACTH. Protocols based upon ELISAs, radioimmunoassays, Western blotting,and the use of BIAcore® analysis are suitable for use in such simplecompetition studies.

In certain embodiments, one would pre-mix the control anti-ACTH antibodywith varying amounts of the test antibody (e.g., in ratios of about 1:1,1:2, 1:10 or about 1:100) for a period of time prior to applying to theACTH antigen sample. In other embodiments, the control and varyingamounts of test antibody can simply be added separately and admixedduring exposure to the ACTH antigen sample. As long as one candistinguish bound from free antibodies (e.g., by using separation orwashing techniques to eliminate unbound antibodies) and control antibodyfrom the test antibody (e.g., by using species specific or isotypespecific secondary antibodies or by specifically labeling the controlantibody with a detectable label) one will be able to determine if thetest antibody reduces the binding of the control antibody to the ACTHantigens, indicating that the test antibody recognizes substantially thesame epitope as the control anti-ACTH antibody. The binding of the(labeled) control antibody in the presence of a completely irrelevantantibody (that does not bind ACTH) can serve as the control high value.The control low value can be obtained by incubating the labeled controlantibody with the same but unlabeled control antibody, where competitionwould occur and reduce binding of the labeled antibody. In a test assay,a significant reduction in labeled antibody reactivity in the presenceof a test antibody is indicative of a test antibody that recognizessubstantially the same epitope, i.e., one that competes with the labeledcontrol antibody. For example, any test antibody that reduces thebinding of the control antibody to ACTH by at least about 50%, such asat least about 60%, or more preferably at least about 70% (e.g., about65-100%), at any ratio of test antibody between about 1:1 or 1:10 andabout 1:100 is considered to be an antibody that binds to substantiallythe same or overlapping epitope or determinant as the control antibody.

Preferably, such test antibody will reduce the binding of the controlantibody to ACTH antigen preferably at least about 50%, at least about60%, at least about 80% or at least about 90% (e.g., about 95%) of thebinding of the control antibody observed in the absence of the testantibody.

A simple competition assay in which a test antibody is pre-adsorbed andapplied at saturating concentration to a surface onto which ACTH isimmobilized also may be advantageously employed. The surface in thesimple competition assay is preferably a BIAcore® chip (or other mediasuitable for surface plasmon resonance analysis). The binding of acontrol antibody that binds ACTH to the ACTH-coated surface is measured.This binding to the ACTH-containing surface of the control antibodyalone is compared with the binding of the control antibody in thepresence of a test antibody. A significant reduction in binding to theACTH-containing surface by the control antibody in the presence of atest antibody indicates that the test antibody recognizes substantiallythe same epitope as the control antibody such that the test antibody“competes” with the control antibody. Any test antibody that reduces thebinding of control antibody by at least about 20% or more, at leastabout 40%, at least about 50%, at least about 70%, or more, can beconsidered to be an antibody that binds to substantially the sameepitope or determinant as the control antibody. Preferably, such testantibody will reduce the binding of the control antibody to ACTH by atleast about 50% (e.g., at least about 60%, at least about 70%, or more).It will be appreciated that the order of control and test antibodies canbe reversed; i.e. the control antibody can be first bound to the surfaceand then the test antibody is brought into contact with the surfacethereafter in a competition assay. Preferably, the antibody havinggreater affinity for ACTH antigen is bound to the ACTH-containingsurface first, as it will be expected that the decrease in binding seenfor the second antibody (assuming the antibodies are competing) will beof greater magnitude. Further examples of such assays are provided ine.g., Saunal and Regenmortel, (1995) J Immunol. Methods 183: 33-41, thedisclosure of which is incorporated herein by reference.

In addition, whether an antibody binds the same or overlappingepitope(s) on ACTH as another antibody or the epitope bound by a testantibody may in particular be determined using a western-blot basedassay. In this assay a library of peptides corresponding to the antigenbound by the antibody, herein ACTH is made, which correspond tooverlapping portions of the protein, typically 10-25, 10-20 or 10-15amino acids long. These different overlapping amino acid peptidesencompassing the ACTH sequence are synthesized and covalently bound to aPepSpots nitrocellulose membrane (JPT Peptide technologies, Berlin,Germany). Blots are then prepared and probed according to themanufacturer's recommendations.

Essentially, the immunoblot assay then detects by fluorometric meanswhat peptides in the library bind to the test antibody and thereby canidentify what residues on the antigen, i.e., ACTH, interact with thetest antibody. (See an embodiment of this technique in U.S. Pat. No.7,935,340, incorporated by reference herein).

The expressions “framework region” or “FR” refer to one or more of theframework regions within the variable regions of the light and heavychains of an antibody (See Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,(1987)). These expressions include those amino acid sequence regionsinterposed between the CDRs within the variable regions of the light andheavy chains of an antibody.

Anti-ACTH Antibodies and Binding Fragments Thereof Having BindingActivity for ACTH

Adrenocorticotropic hormone (ACTH), also known as corticotropin, is apolypeptide tropic hormone produced and secreted by the anteriorpituitary gland. It is an important component of thehypothalamic-pituitary-adrenal axis and is often produced in response tobiological stress (along with its precursor corticotropin-releasinghormone from the hypothalamus). Its principal effects are increasedproduction and release of corticosteroids. When a pituitary tumor is thecause of elevated ACTH (from the anterior pituitary) this is known asCushing's Disease and the constellation of signs and symptoms of theexcess cortisol (hypercortisolism) is known as Cushing's Syndrome. Adeficiency of ACTH is a cause of secondary adrenal insufficiency. ACTHis also related to the circadian rhythm in many organisms. Moreover,elevated ACTH and cortisol production have been associated with sleepapnea, particularly OSA. See Henley et al., J. Clin Endocrinol Metab.November 2009, 94(11):4234-4242.

POMC, ACTH and β-lipotropin are secreted from corticotropes in theanterior lobe (or adenohypophysis) of the pituitary gland in response tothe hormone corticotropin-releasing hormone (CRH) released by thehypothalamus. ACTH is synthesized from pre-pro-opiomelanocortin(pre-POMC). The removal of the signal peptide during translationproduces the 241-amino acid polypeptide POMC, which undergoes a seriesof post-translational modifications such as phosphorylation andglycosylation before it is proteolytically cleaved by endopeptidases toyield various polypeptide fragments with varying physiological activity.

ACTH consists of 39 amino acids and can be processed into two shorterpeptides, α-melanocyte-stimulating hormone (α-MSH) and CLIP. Alpha-MSHconsists of amino acids 1-13 of human ACTH and CLIP consists of aminoacids 18-39 of human ACTH. Human ACTH has a molecular weight of 4,540atomic mass units (Da).

ACTH stimulates secretion of glucocorticoid steroid hormones fromadrenal cortex cells, especially in the zona fasciculata of the adrenalglands. ACTH acts by binding to cell surface ACTH receptors, e.g., MC2R,which are located primarily on adrenocortical cells of the adrenalcortex. The ACTH receptor is a seven-membrane-spanning G protein-coupledreceptor. Upon ligand binding, the receptor undergoes conformationchanges that stimulate the enzyme adenylyl cyclase, which leads to anincrease in intracellular cAMP and subsequent activation of proteinkinase A.

ACTH influences steroid hormone secretion by both rapid short-termmechanisms that take place within minutes and slower long-term actions.The rapid actions of ACTH include stimulation of cholesterol delivery tothe mitochondria where the P450scc enzyme is located. P450scc catalyzesthe first step of steroidogenesis that is cleavage of the side-chain ofcholesterol. ACTH also stimulates lipoprotein uptake into corticalcells. This increases the bio-availability of cholesterol in the cellsof the adrenal cortex.

The long term actions of ACTH include stimulation of the transcriptionof the genes coding for steroidogenic enzymes, especially P450scc,steroid 11β-hydroxylase, and their associated electron transferproteins. This effect is observed over several hours.

The present invention provides novel antibodies or antibody fragmentsthat bind ACTH, including human ACTH. In preferred embodiments, theantibody or antibody fragment according to the invention comprises oneor more complementarity determining regions (CDRs) of the anti-ACTHantibodies and antibody fragments described herein.

In some embodiments, an anti-ACTH antibody or antibody fragmentaccording to the invention will interfere with, block, reduce ormodulate the interaction between ACTH and MCRs (e.g., MC1R, MC2R, MC3R,MC4R and/or MC5R). In some instances an anti-ACTH antibody or antibodyfragment according to the invention is denoted as “neutralizing”, e.g.,if it totally prevents the interaction of ACTH and MCR. In someembodiments, the antibody or antibody fragment neutralizes ACTH, e.g.,by remaining bound to ACTH in a location and/or manner that preventsACTH from binding to MCRs. This in turn results in a reduction in theamount of serum cortisol present in a subject.

In some embodiments, the antibody or antibody fragment according to theinvention are capable of inhibiting ACTH-mediated activity (includingbinding). In some embodiments, the antibody or antibody fragmentaccording to the invention are humanized, such as humanized rabbitantibodies to ACTH.

As mentioned, the anti-ACTH antibodies or antibody fragments accordingto the invention have a variety of utilities. For example, the subjectantibodies and fragments are useful in therapeutic applications, as wellas diagnostically in binding assays, and are useful for affinitypurification of ACTH, in particular human ACTH or its ligands and inscreening assays to identify other antagonists of ACTH activity. Some ofthe antibodies or antibody fragments according to the invention areuseful for inhibiting binding of ACTH to MCRs, or inhibitingACTH-mediated activities.

The antibody or antibody fragment according to the invention can be usedin a variety of therapeutic applications. For example, in someembodiments the anti-ACTH antibody or antibody fragment according to theinvention are useful for treating conditions associated with ACTH, suchas congenital adrenal hyperplasia (CAH), Classical CAH, NonclassicalCAH, familial glucocorticoid deficiency (FGD), Cushing's Disease,Cushing's Syndrome, obesity, diabetes, depression, anxiety disorders,cancer (such as Cushing's Syndrome resulting from ectopic ACTHexpression, e.g., in small cell lung cancer, non-small cell lung cancer(NSCLC), pancreatic carcinoma, neural tumors, or thymoma), muscleatrophy, hypertension, sleep apnea, hyperinsulinemia, cognitivedysfunction, Alzheimer's disease, galactorrhea, stress relatedconditions, impaired cardiac function, exercise intolerance, heartfailure and other cardiac conditions, metabolic syndrome, andhyperaldosteronism including primary hyperaldosteronism (such as Conn'ssyndrome), secondary hyperaldosteronism, and familialhyperaldosteronism, and other diseases, disorders, and conditions.

The subject anti-ACTH antibodies and antibody fragments according to theinvention can in particular be used for treating any subject whereinblocking, inhibiting or neutralizing the in vivo effect of ACTH orblocking or inhibiting the interaction of ACTH and MCRs istherapeutically desirable, wherein the subject anti-ACTH antibodies orantibody fragments may be used alone or in association with other activeagents or drugs.

Said treatment may include administration of another agent. Exemplaryagents may be agents used for the treatment of a condition associatedwith ACTH, such as congenital adrenal hyperplasia (CAH), Classical CAH,Nonclassical CAH, familial glucocorticoid deficiency (FGD), ACTH-drivenhypercortisolism, acute coronary syndrome, acute heart failure, anxietydisorders, atherosclerosis, atrial fibrillation, cachexia, cancer (suchas Cushing's Syndrome resulting from ectopic ACTH expression, e.g., insmall cell lung cancer, non-small cell lung cancer (NSCLC), pancreaticcarcinoma, neural tumors, or thymoma), cardiac conditions, cardiacfibrosis, cardiovascular disorders, chronic renal failure, chronicstress syndrome, cognitive dysfunction, Alzheimer's disease, congestiveheart failure, Conn's syndrome, coronary heart diseases, Cushing'sDisease, Cushing's Syndrome, depression, diabetes, endothelialdysfunction, exercise intolerance, familial hyperaldosteronism,fibrosis, galactorrhea, heart failure, hyperaldosteronism,hypercortisolemia, hypertension, hypokalemia, impaired cardiac function,increased formation of collagen, inflammation, metabolic syndrome,muscle atrophy, conditions associated with muscle atrophy, myocardiacfibrosis, nephropathy, obesity, post-myocardial infarction, primaryhyperaldosteronism, remodeling following hypertension, renal failure,restenosis, secondary hyperaldosteronism, sleep apnea, or syndrome X, orfor the treatment of a related condition such as hypercholesterolemia.

Additional exemplary agents that may be administered include (i)angiotensin II receptor antagonist or a pharmaceutically acceptable saltthereof, (ii) HMG-Co-A reductase inhibitor or a pharmaceuticallyacceptable salt thereof, (iii) angiotensin converting enzyme (ACE)Inhibitor or a pharmaceutically acceptable salt thereof, (iv) calciumchannel blocker (CCB) or a pharmaceutically acceptable salt thereof, (v)dual angiotensin converting enzyme/neutral endopeptidase (ACE/NEP)inhibitor or a pharmaceutically acceptable salt thereof, (vi) endothelinantagonist or a pharmaceutically acceptable salt thereof, (vii) renininhibitor or a pharmaceutically acceptable salt thereof, (viii) diureticor a pharmaceutically acceptable salt thereof, (ix) an ApoA-1 mimic; (x)an anti-diabetic agent; (xi) an obesity-reducing agent; (xii) analdosterone receptor blocker; (xiii) an endothelin receptor blocker;(xiv) a CETP inhibitor; (xv) an inhibitor of Na—K-ATPase membrane pump;(xvi) a beta-adrenergic receptor blocker or an alpha-adrenergic receptorblocker; and (xvii) a neutral endopeptidase (NEP) inhibitor; or anycombination thereof.

Further non-limiting examples of drugs that may be co-administered withthe subject antibodies or antibody fragments or used in the sametherapeutic regimen include by way of example statins, ACE inhibitors,Angiotensin II receptor blockers (ARBs), antiarrhythmics, antiplateletdrugs, aspirin, beta blockers, amiodarone, digoxin, aspirin,anti-clotting agents, digoxin, diuretics, heart failure drugs,vasodilators, blood thinners, other anti-cholesterol drugs such asholestyramine (Questran), gemfibrozil (Lopid, Gemcor), Omacor, andpantethine, other anti-hypertensives, antidiabetogenic drugs such asalpha-glucosidase inhibitors, biguanides, dipeptidyl peptidase-4inhibitors, insulin therapies, meglitinides, sulfonylurea, andthiazolidinediones, and other drugs used to treat hypertension andconditions that are frequently associated with hypertension (such ashypercholesterolemia, diabetes, metabolic syndrome, obesity, etc.).

ACE inhibitors may be used in combination with the subject anti-ACTHantibodies and antibody fragments wherein the moieties may be jointly orseparately administered by the same or different means of administrationinclude by way of example: Capoten (captopril), Vasotec (enalapril),Prinivil, Zestril (lisinopril), Lotensin (benazepril), Monopril(fosinopril), Altace (ramipril), Accupril (quinapril), Aceon(perindopril), Mavik (trandolapril), and Univasc (moexipril) as well asany pharmaceutically acceptable salts thereof.

ARBs may be used in combination with the subject anti-ACTH antibodiesand antibody fragments wherein the moieties may be jointly or separatelyadministered by the same or different means of administration include byway of example: Cozaar (losartan), Diovan (valsartan), Avapro(irbesartan), Atacand (candesartan), Micardis (telmisartan), eprosartan,olmesartan, saprisartan, tasosartan, E-4177, SC-52458, and ZD8731, aswell as any pharmaceutically acceptable salts thereof.

Antiarrhythmics may be used in combination with the subject anti-ACTHantibodies and antibody fragments include by way of example: Tambocor(flecainide), Procanbid (procainamide), Cordarone (amiodarone), andBetapace (sotalol).

Anticlotting agents which may be used in combination with the subjectanti-ACTH antibodies and antibody fragments wherein the moieties may bejointly or separately administered by the same or different means ofadministration include: Tissue plasminogen activator (TPA),Tenecteplase, Alteplase, Urokinase, Reteplase, and Streptokinase.

Beta-blockers may be used in combination with the subject anti-ACTHantibodies and antibody fragments wherein the agents may be jointly orseparately administered by the same or different means of administrationinclude by way of example: Sectral (acebutolol), Zebeta (bisoprolol),Brevibloc (esmolol), Inderal (propranolol), Tenormin (atenolol),Normodyne, Trandate (labetalol), Coreg (carvedilol), Lopressor, andToprol-XL (metoprolol).

Calcium channel blockers which may be used in combination with thesubject anti-ACTH antibodies and antibody fragments wherein the agentsmay be jointly or separately administered by the same or different meansof administration include by way of example: Norvasc (amlodipine),Plendil (felodipine), Cardizem, Cardizem CD, Cardizem SR, Dilacor XR,Diltia XT, Tiazac (diltiazem), Calan, Calan SR, Covera-HS, Isoptin,Isoptin SR, Verelan, Verelan PM (verapamil), Adalat, Adalat CC,Procardia, Procardia XL (nifedipine), Cardene, Cardene SR (nicardipine),Sular (nisoldipine), Vascor (bepridil), and Caduet which is acombination of a statin cholesterol drug and amlodipine.

Diuretics which may be used in combination with the subject anti-ACTHantibodies and antibody fragments wherein the agents may be jointly orseparately administered by the same or different means of administrationinclude by way of example Lasix (furosemide), Bumex (bumetanide),Demadex (torsemide), Esidrix (hydrochlorothiazide), Zaroxolyn(metolazone), Aldactone (spironolactone), ethacrynic acid, ethynacrylicacid, mersalyl with theophylline, mercaptomerin sodium, merethoxyllineprocaine, amiloride, triamterene, chlorothalidone, chlorothiazide,quinethazone, hydroflumethiazide, methylchlorothiazide, anddichlorphenamide, including any pharmaceutically acceptable saltsthereof.

Heart failure drugs which may be used in combination with the subjectanti-ACTH antibodies and antibody fragments wherein the agents may bejointly or separately administered by the same or different means ofadministration include by way of example Dobutrex (dobutamine), andPrimacor (milrinone).

Vasodilators which may be used in combination with the subject anti-ACTHantibodies and antibody fragments wherein the agents may be jointly orseparately administered by the same or different means of administrationinclude by way of example Dilatrate-SR, Iso-Bid, Isonate, Isorbid(isosorbide dinitrate), Isordil, Isotrate, Sorbitrate (isosorbidedinitrate), IMDUR (isosorbide mononitrate), and BiDil (hydralazine withisosorbide dinitrate.

Blood thinners which may be used in combination with the subjectanti-ACTH antibodies and antibody fragments wherein the agents may bejointly or separately administered by the same or different means ofadministration include by way of example warfarin (Coumadin), Heparin,Lovenox, and Fragmin.

The subject anti-ACTH antibodies and antibody fragments according to theinvention can further in particular be used for treating any subjectwherein reducing cortisol and/or corticosterone levels isprophylactically or therapeutically desirable, wherein the subjectanti-ACTH antibodies or antibody fragments may be used alone or inassociation with other active agents or drugs. These conditions includeby way of example Cushing's Disease, Cushing's Syndrome, obesity,diabetes, sleep apnea, depression, anxiety disorders, cancer (such asCushing's Syndrome resulting from ectopic ACTH expression, e.g., insmall cell lung cancer, non-small cell lung cancer (NSCLC), pancreaticcarcinoma, neural tumors, or thymoma), muscle atrophy, hypertension,hyperinsulinemia, cognitive dysfunction, Alzheimer's disease,galactorrhea, stress related conditions, impaired cardiac function,exercise intolerance, heart failure and other cardiac conditions,metabolic syndrome, hyperaldosteronism including primaryhyperaldosteronism (such as Conn's syndrome) secondaryhyperaldosteronism, and familial hyperaldosteronism, and other diseases,disorders, and conditions.

The subject anti-ACTH antibodies and antibody fragments according to theinvention can also be used in any of the aforementioned therapeuticindications or conditions in combination with other drugs that aretypically used to treat such disorders, wherein the antibody and otherdrug or agent may be co-administered or separately administered.

In particular, there are several pharmacological approaches to thetreatment of Cushing's disease and/or Cushing's Syndrome. Drugs used tosuppress cortisol secretion are mostly inhibitors of steroidogenesis,including, but not limited to, ketoconazole (Nizoral®),aminoglutethimide (Cytadren®), metyrapone (Metopirone®), mitotane(Lysodren®) and etomidate (Amidate®). Drugs that suppressadrenocorticotropic hormone (ACTH) secretion, e.g., cyproheptadine(Periactin® or Peritol®), valproic acid (Depakote®), cabergoline(Dostinex®), somatostatin analogs (e.g., pasireotide (Signifor®)),PPAR-gamma agonists (e.g., rosiglitazone (Avandia®)), vasopressinantagonists (i.e., Vaptans, including, but not limited to, conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), and satavaptan(SR121463, planned trade name Aquilda®)), may also be used. A thirdcategory of drugs is glucocorticoid receptor antagonists, e.g.,mifepristone (Korlym®).

As noted above, the subject anti-ACTH antibodies may be used for theprevention or treatment of diseases and conditions associated withelevated aldosterone, and/or diseases and conditions treatable bydecreasing aldosterone. Said diseases and conditions includehypertension, cardiovascular disorders, impaired cardiac function,exercise intolerance, heart failure (including congestive heart failureand acute heart failure), cardiac conditions, hypokalemia, atrialfibrillation, renal failure (e.g., chronic renal failure), restenosis,sleep apnea, atherosclerosis, syndrome X, obesity, nephropathy,post-myocardial infarction, coronary heart diseases, inflammation,increased formation of collagen, fibrosis such as cardiac or myocardiacfibrosis and remodeling following hypertension, endothelial dysfunction,cachexia, acute coronary syndrome, chronic stress syndrome, Cushing'sdisease, Cushing's Syndrome, metabolic syndrome, hypercortisolemia, andhyperaldosteronism (including primary hyperaldosteronism, secondaryhyperaldosteronism, and familial hyperaldosteronism).

Additionally, there are several approaches to the management and/ortreatment of sleep disorders, such as sleep apnea, insomnia ornarcolepsy, ranging from lifestyle changes, such as losing weight orquitting smoking, to supplemental oxygen, medical devices, surgeryand/or pharmaceuticals such as antidepressants and other drugs. Usingsupplemental oxygen while you sleep may treat sleep apnea. Various formsof oxygen are available as well as different devices to deliver oxygento your lungs. Exemplary therapies include, but are not limited to,continuous positive airway pressure (CPAP); adjustable airway pressuredevices (e.g., BPAP); expiratory positive airway pressure (EPAP); andoral appliances. CPAP therapy uses a machine to deliver air pressure,which is somewhat greater than that of the surrounding air, to keep yourupper airway passages open, preventing apnea and snoring. Adjustableairway pressure devices provide an automatically adjusted air pressureto a subject while sleeping. For example, bilevel positive airwaypressure (BPAP) therapy used a device that provides more pressure whenyou inhale and less when you exhale. EPAP is a small, single-use devicethat is placed over each nostril before going to sleep. The device is avalve that allows air to move freely in, but when you exhale, air mustgo through small holes in the valve which increases pressure in theairway and keeps it open. Also, adaptive servo-ventilation (ASV) is anairflow device that “learns” a person's normal breathing pattern andstores the information in a built-in computer so that after fallingasleep, the machine uses pressure to normalize the breathing pattern andprevent pauses in your breathing. Another option is wearing an oralappliance designed to keep your throat open, e.g., by bringing your jawforward. Additionally, surgical intervention (i.e., to enlarge theairway through your nose or throat) is another approach to the treatmentof sleep apnea. Exemplary surgical options include, but are not limitedto, tissue removal (i.e., uvulopalatoparyngoharyngoplasty (UPPP) and/orremoval of tonsils and adenoids); jaw repositioning (i.e.,maxillomandibular advancement); implants (e.g., implanting plastic rodsinto the soft palate); creating a new air passageway (i.e.,tracheostomy); nasal surgery to remove polyps or straighten a crookedpartition between your nostrils (e.g., deviated nasal septum); andsurgery to remove enlarged tonsils or adenoids. Additionally, treatingmedical problems associated with sleep apnea, e.g., heart orneuromuscular disorders, may improve and/or eliminate the symptoms ofcentral sleep apnea. Finally, drugs used to treat sleep apnea include,but are not limited to, armodafinil (Nuvigil®) and modafinil(Provigil®).

Examples of drugs that may be co-administered with the subject anti-ACTHantibodies or antibody fragments or in the same therapeutic regimeninclude, by way of example, ketoconazole (Nizoral®), aminoglutethimide(Cytadren®), metyrapone (Metopirone®), mitotane (Lysodren®) etomidate(Amidate®), cyproheptadine (Periactin® or Peritol®), valproic acid(Depakote®), cabergoline (Dostinex®), pasireotide (Signifor®),rosiglitazone (Avandia®), conivaptan (Vaprisol®), tolvaptan (OPC-41061),lixivaptan (VPA-985), satavaptan (SR121463, planned trade nameAquilda®), mifepristone (Korlym®), armodafinil (Nuvigil®) and modafinil(Provigil®), and other drugs used to treat conditions wherein thetreated individual may have elevated ACTH levels. Further, examples ofdrugs that may be co-administered with the subject anti-ACTH antibodiesor antibody fragments or in the same therapeutic regimen include withoutlimitation thereto one or more of: Accupril (quinapril), Aceon(perindopril), Adalat, Adalat CC, Aldactone (spironolactone),aldosterone receptor blockers, alpha-adrenergic receptor blockers,alpha-glucosidase inhibitors, Altace (ramipril), Alteplase,aminoglutethimide (Cytadren®), amiodarone, angiotensin converting enzyme(ACE) Inhibitors, angiotensin II receptor antagonists, Angiotensin IIreceptor blockers (ARBs), antiarrhythmics, anti-cholesterol drugs,anti-clotting agents, antidiabetogenic drugs, anti-hypertensive agents,antiplatelet drugs, ApoA-1 mimics, aspirin, Atacand (candesartan),Avapro (irbesartan), beta blockers, beta-adrenergic receptor blockers,Betapace (sotalol), BiDil (hydralazine with isosorbide dinitrate),biguanides, blood thinners, Brevibloc (esmolol), Bumex (bumetanide),cabergoline (Dostinex®), Caduet (a combination of a statin cholesteroldrug and amlodipine), Calan, Calan SR, Calcium channel blockers, Capoten(captopril), Cardene, Cardene SR (nicardipine), Cardizem, Cardizem CD,Cardizem SR, CETP inhibitors, conivaptan (Vaprisol®), Cordarone(amiodarone), Coreg (carvedilol), Covera-HS, Cozaar (losartan),cyproheptadine (Periactin® or Peritol®), Demadex (torsemide), digoxin,Dilacor XR, Dilatrate-SR, Diltia XT, Diovan (valsartan), dipeptidylpeptidase-4 inhibitors, diuretics, Dobutrex (dobutamine), drugs thatsuppress ACTH secretion, drugs that suppress cortisol secretion, dualangiotensin converting enzyme/neutral endopeptidase (ACE/NEP)inhibitors, endothelin antagonists, endothelin receptor blockers,Esidrix (hydrochlorothiazide), etomidate (Amidate®), Fragmin,gemfibrozil (Lopid, Gemcor), glucocorticoid receptor antagonists, heartfailure drugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine(Questran), IMDUR (isosorbide mononitrate), Inderal (propranolol),inhibitors of a Na—K-ATPase membrane pump, inhibitors ofsteroidogenesis, insulin therapies, Iso-Bid, Isonate, Isoptin, IsoptinSR, Isorbid (isosorbide dinitrate), Isordil, Isotrate, ketoconazole(Nizoral®), Lasix (furosemide), lixivaptan (VPA-985), Lopressor,Lotensin (benazepril), Lovenox, Mavik (trandolapril), meglitinides,metyrapone (Metopirone®), Micardis (telmisartan), mifepristone(Korlym®), mitotane (Lysodren®), Monopril (fosinopril), neutralendopeptidase (NEP) inhibitors, Normodyne, Norvasc (amlodipine),obesity-reducing agents, Omacor, pantethine, pasireotide (Signifor®),Plendil (felodipine), PPAR-gamma agonists, Primacor (milrinone),Prinivil, Procanbid (procainamide), Procardia, Procardia XL(nifedipine), renin inhibitors, Reteplase, rosiglitazone (Avandia®),satavaptan (SR121463, planned trade name Aquilda®), Sectral(acebutolol), somatostatin analogs, Sorbitrate (isosorbide dinitrate),statins, Streptokinase, Sular (nisoldipine), sulfonylurea, Tambocor(flecainide), Tenecteplase, Tenormin (atenolol), thiazolidinediones,Tiazac (diltiazem), Tissue plasminogen activator (TPA), tolvaptan(OPC-41061), Toprol-XL (metoprolol), Trandate (labetalol), Univasc(moexipril), Urokinase, valproic acid (Depakote®), vaptans, Vascor(bepridil), vasodilators, Vasodilators, vasopressin antagonists, Vasotec(enalapril), Verelan, Verelan PM (verapamil), warfarin (Coumadin),Zaroxolyn (metolazone), Zebeta (bisoprolol), or Zestril (lisinopril).Further exemplary active agents include one or more corticosteroids,including glucocorticoids and/or mineralocorticoids (including agentshaving one or both of glucocorticoid and/or mineralocorticoid activity),such as cortisol (hydrocortisone), dexamethasone, cortisone, prednisone,prednisolone, methylprednisolone, betamethasone, triamcinolone,beclometasone, fludrocortisone (e.g., fludrocortisone acetate),deoxycorticosterone (e.g., deoxycorticosterone acetate (DOCA)), and/oraldosterone.

It should also be noted that the anti-ACTH antibodies or antibodyfragments of the present invention may be used in conjunction with anyof the described non-pharmaceutical based therapies for sleep apnea.Accordingly, in one embodiment, the anti-ACTH antibodies or antibodyfragments are used in combination with one or more of lifestyle changes,supplemental oxygen, medical devices, and surgery to treat sleep apnea.

The invention further relates to compositions containing the subjectanti-ACTH antibodies or antibody fragments, especially compositions aresuitable for in vivo administration, e.g., subcutaneous, intravenous,intradermal, intranasal, intrathecal, vaginal, rectal, and otherinjectable administrable dosage forms.

More specifically, the invention provides compositions containing thesubject anti-ACTH antibodies or antibody fragments, especiallycompositions which are suitable for in vivo administration, e.g.,subcutaneous, intravenous, intradermal, intranasal, intrathecal,vaginal, rectal, oral and other injectable dosage forms which optionallymay contain another active agent such as ketoconazole (Nizoral®),aminoglutethimide (Cytadren®), metyrapone (Metopirone®), mitotane(Lysodren®) etomidate (Amidate®), cyproheptadine (Periactin® orPeritol®), valproic acid (Depakote®), cabergoline (Dostinex®),pasireotide (Signifor®), rosiglitazone (Avandia®), conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), satavaptan(SR121463, planned trade name Aquilda®), mifepristone (Korlym®),armodafinil (Nuvigil®) and modafinil (Provigil®), and other drugs usedto treat conditions wherein the treated individual may have elevatedACTH levels. Further examples of other active agent(s) that mayoptionally be contained in said dosage form include without limitationthereto one or more of: Accupril (quinapril), Aceon (perindopril),Adalat, Adalat CC, Aldactone (spironolactone), aldosterone receptorblockers, alpha-adrenergic receptor blockers, alpha-glucosidaseinhibitors, Altace (ramipril), Alteplase, aminoglutethimide (Cytadren®),amiodarone, angiotensin converting enzyme (ACE) Inhibitors, angiotensinII receptor antagonists, Angiotensin II receptor blockers (ARBs),antiarrhythmics, anti-cholesterol drugs, anti-clotting agents,antidiabetogenic drugs, anti-hypertensive agents, antiplatelet drugs,ApoA-1 mimics, aspirin, Atacand (candesartan), Avapro (irbesartan), betablockers, beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR, CETPinhibitors, conivaptan (Vaprisol®), Cordarone (amiodarone), Coreg(carvedilol), Covera-HS, Cozaar (losartan), cyproheptadine (Periactin®or Peritol®), Demadex (torsemide), digoxin, Dilacor XR, Dilatrate-SR,Diltia XT, Diovan (valsartan), dipeptidyl peptidase-4 inhibitors,diuretics, Dobutrex (dobutamine), drugs that suppress ACTH secretion,drugs that suppress cortisol secretion, dual angiotensin convertingenzyme/neutral endopeptidase (ACE/NEP) inhibitors, endothelinantagonists, endothelin receptor blockers, Esidrix(hydrochlorothiazide), etomidate (Amidate®), Fragmin, gemfibrozil(Lopid, Gemcor), glucocorticoid receptor antagonists, heart failuredrugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine (Questran),IMDUR (isosorbide mononitrate), Inderal (propranolol), inhibitors of aNa—K-ATPase membrane pump, inhibitors of steroidogenesis, insulintherapies, Iso-Bid, Isonate, Isoptin, Isoptin SR, Isorbid (isosorbidedinitrate), Isordil, Isotrate, ketoconazole (Nizoral®), Lasix(furosemide), lixivaptan (VPA-985), Lopressor, Lotensin (benazepril),Lovenox, Mavik (trandolapril), meglitinides, metyrapone (Metopirone®),Micardis (telmisartan), mifepristone (Korlym®), mitotane (Lysodren®),Monopril (fosinopril), neutral endopeptidase (NEP) inhibitors,Normodyne, Norvasc (amlodipine), obesity-reducing agents, Omacor,pantethine, pasireotide (Signifor®), Plendil (felodipine), PPAR-gammaagonists, Primacor (milrinone), Prinivil, Procanbid (procainamide),Procardia, Procardia XL (nifedipine), renin inhibitors, Reteplase,rosiglitazone (Avandia®), satavaptan (SR121463, planned trade nameAquilda®), Sectral (acebutolol), somatostatin analogs, Sorbitrate(isosorbide dinitrate), statins, Streptokinase, Sular (nisoldipine),sulfonylurea, Tambocor (flecainide), Tenecteplase, Tenormin (atenolol),thiazolidinediones, Tiazac (diltiazem), Tissue plasminogen activator(TPA), tolvaptan (OPC-41061), Toprol-XL (metoprolol), Trandate(labetalol), Univasc (moexipril), Urokinase, valproic acid (Depakote®),vaptans, Vascor (bepridil), vasodilators, Vasodilators, vasopressinantagonists, Vasotec (enalapril), Verelan, Verelan PM (verapamil),warfarin (Coumadin), Zaroxolyn (metolazone), Zebeta (bisoprolol), orZestril (lisinopril). Further exemplary active agents include one ormore corticosteroids, including glucocorticoids and/ormineralocorticoids (including agents having one or both ofglucocorticoid and/or mineralocorticoid activity), such as cortisol(hydrocortisone), dexamethasone, cortisone, prednisone, prednisolone,methylprednisolone, betamethasone, triamcinolone, beclometasone,fludrocortisone (e.g., fludrocortisone acetate), deoxycorticosterone(e.g., deoxycorticosterone acetate (DOCA)), and/or aldosterone.

The invention also provides novel dosage regimens using the subjectanti-ACTH antibodies or antibody fragments, alone or in association withanother active, especially subcutaneous, oral and intravenous dosingregimens.

Other uses for the antibodies or antibody fragments according to theinvention include, for example, diagnosis of ACTH-associated diseases orconditions and screening assays to determine the presence or absence ofACTH. Some of the antibodies or antibody fragments according to theinvention described herein are useful in treating consequences,symptoms, and/or the pathology associated with ACTH activity.

Exemplary anti-ACTH antibodies and antibody fragments according to theinvention, and the specific CDRs thereof are identified in the followingsection. For the reader's convenience, each exemplified antibody orfragment, and sequences contained therein, are separately describedunder a Header that identifies the exemplified antibody by a specificnomenclature, i.e., Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H, preferably Ab13.H.

Antibody Polypeptide Sequences

Antibody Ab13

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 1) QQLEESGGGLVKPGGTLTLTCTASGFSFSSGYDICWARQGPGKGLEWIGCIDTGSGNTYYASWAKGRFTMSRTSSTTVTLQVTSLTAADTATYFCAKGISSIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 2) QQLEESGGGLVKPGGTLTLTCTASGFSFSSGYDICWARQGPGKGLEWIGCIDTGSGNTYYASWAKGRFTMSRTSSTTVTLQVTSLTAADTATYFCAKGIS SIWGPGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab13 and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 10) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 21) DIVMTQTPASVSEPVGGTVTIKCQASQTISSDLAWYQQKPGQPPKLLIYAASKLTSGVSSRFKGGGTGTQFTLTISDLECADAATYYCQTYYDIIDDGCTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 22) DIVMTQTPASVSEPVGGTVTIKCQASQTISSDLAWYQQKPGQPPKLLIYAASKLTSGVSSRFKGGGTGTQFTLTISDLECADAATYYCQTYYDIIDDGCT FGGGTEVVVKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab13 which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 30) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 4; SEQ ID NO: 6; andSEQ ID NO: 8 which correspond to the complementarity-determining regions(CDRs, or hypervariable regions) of the heavy chain sequence of SEQ IDNO: 1 or which contain the variable heavy chain sequence of SEQ ID NO:2, and/or which further contain one, two, or three of the polypeptidesequences of SEQ ID NO: 24; SEQ ID NO: 26; and SEQ ID NO: 28 whichcorrespond to the complementarity-determining regions (CDRs, orhypervariable regions) of the light chain sequence of SEQ ID NO: 21 orwhich contain the variable light chain sequence of SEQ ID NO: 22, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9which correspond to the framework regions (FRs or constant regions) ofthe heavy chain sequence of SEQ ID NO: 1 or the variable heavy chainsequence of SEQ ID NO: 2, and/or one, two, three, or four of thepolypeptide sequences of SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27;and SEQ ID NO: 29 which correspond to the framework regions (FRs orconstant regions) of the light chain sequence of SEQ ID NO: 21 or thevariable light chain sequence of SEQ ID NO: 22, or combinations of thesepolypeptide sequences or sequences which are at least 80%, 90%, 95%,96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 21 or SEQ ID NO: 22 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 4; SEQ ID NO: 6; and SEQ ID NO: 8 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 1 or the variable heavy chainsequence of SEQ ID NO: 2 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 24; SEQ ID NO: 26; and SEQ ID NO: 28 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 21 or the variable light chainsequence of SEQ ID NO: 22 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; and SEQ ID NO: 9 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 1 or the variable heavy chainsequence of SEQ ID NO: 2 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 23; SEQ ID NO: 25; SEQ ID NO: 27; and SEQ ID NO:29 which correspond to the framework regions (FRs or constant regions)of the light chain sequence of SEQ ID NO: 21 or the variable light chainsequence of SEQ ID NO: 22 or sequences that are at least 90% or 95%identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 2; the variable lightchain region of SEQ ID NO: 22; the complementarity-determining regions(SEQ ID NO: 4; SEQ ID NO: 6; and SEQ ID NO: 8) of the variable heavychain region of SEQ ID NO: 2; and the complementarity-determiningregions (SEQ ID NO: 24; SEQ ID NO: 26; and SEQ ID NO: 28) of thevariable light chain region of SEQ ID NO: 22 or sequences that are atleast 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 2; the variable light chainregion of SEQ ID NO: 22; the framework regions (SEQ ID NO: 3; SEQ ID NO:5; SEQ ID NO: 7; and SEQ ID NO: 9) of the variable heavy chain region ofSEQ ID NO: 2; and the framework regions (SEQ ID NO: 23; SEQ ID NO: 25;SEQ ID NO: 27; and SEQ ID NO: 29) of the variable light chain region ofSEQ ID NO: 22.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab13, comprising, or alternatively consisting of, SEQ ID NO:1 and SEQ ID NO: 21 or SEQ ID NO: 2 and SEQ ID NO: 22, or an antibody orantibody fragment comprising the CDRs of Ab13 and having at least one ofthe biological activities set forth herein or is an anti-ACTH antibodythat competes with Ab13 in binding ACTH, preferably one containingsequences that are at least 90%, 95%, 96%, 97%, 98% or 99% identical tothat of Ab13 or an antibody that binds to the same or overlappingepitope(s) on ACTH as Ab13.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab13, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 2 and the variable lightchain sequence of SEQ ID NO: 22 or sequences that are at least 90%, 95%,96%, 97%, 98% or 99% identical thereto. This embodiment of the inventionfurther includes Fabs containing additions, deletions, and variants ofSEQ ID NO: 2 and/or SEQ ID NO: 22 which retain the binding specificityfor ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) of Ab13.In another embodiment of the invention, anti-ACTH antibodies such asAb13 or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab13 aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab15

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 41) QQQLEESGGGLVKPGGTLTLTCKGSGIAFSDTYDMCWVRQAPGKGLEWIGCIDTGSGDTYYPTWAKGRFTISKPSSTTVDLKMTSLTAADTATYFCAKGVSSLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 42) QQQLEESGGGLVKPGGTLTLTCKGSGIAFSDTYDMCWVRQAPGKGLEWIGCIDTGSGDTYYPTWAKGRFTISKPSSTTVDLKMTSLTAADTATYFCAKGV SSLWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab15 and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 50) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 61) DIVMTQTPASVSEPVGGTVTIKCQASEDIESDLAWYQQKPGQPPKWYGASTLKSGVSSRFRGSGSGTEYTLTISDLECADAATYYCQTYYDMADDGCSFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 62) DIVMTQTPASVSEPVGGTVTIKCQASEDIESDLAWYQQKPGQPPKLLIYGASTLKSGVSSRFRGSGSGTEYTLTISDLECADAATYYCQTYYDMADDGCS FGGGTEVVVKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab15 which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 70) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 44; SEQ ID NO: 46; andSEQ ID NO: 48 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 41 or which contain the variable heavy chain sequence of SEQID NO: 42, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 64; SEQ ID NO: 66; and SEQ ID NO: 68which correspond to the complementarity-determining regions (CDRs, orhypervariable regions) of the light chain sequence of SEQ ID NO: 61 orwhich contain the variable light chain sequence of SEQ ID NO: 62, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 43; SEQ ID NO: 45; SEQ ID NO: 47; and SEQ ID NO:49 which correspond to the framework regions (FRs or constant regions)of the heavy chain sequence of SEQ ID NO: 41 or the variable heavy chainsequence of SEQ ID NO: 42, and/or one, two, three, or four of thepolypeptide sequences of SEQ ID NO: 63; SEQ ID NO: 65; SEQ ID NO: 67;and SEQ ID NO: 69 which correspond to the framework regions (FRs orconstant regions) of the light chain sequence of SEQ ID NO: 61 or thevariable light chain sequence of SEQ ID NO: 62, or combinations of thesepolypeptide sequences or sequences which are at least 80%, 90%, 95%,96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 41 or SEQ ID NO: 42 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 61 or SEQ ID NO: 62 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 44; SEQ ID NO: 46; and SEQ ID NO: 48 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 41 or the variable heavy chainsequence of SEQ ID NO: 42 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 64; SEQ ID NO: 66; and SEQ ID NO: 68 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 61 or the variable light chainsequence of SEQ ID NO: 62 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 43; SEQ ID NO: 45; SEQ ID NO: 47; and SEQ ID NO: 49 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 41 or the variable heavy chainsequence of SEQ ID NO: 42 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 63; SEQ ID NO: 65; SEQ ID NO: 67; and SEQ ID NO:69 which correspond to the framework regions (FRs or constant regions)of the light chain sequence of SEQ ID NO: 61 or the variable light chainsequence of SEQ ID NO: 62 or sequences that are at least 90% or 95%identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 42; the variable lightchain region of SEQ ID NO: 62; the complementarity-determining regions(SEQ ID NO: 44; SEQ ID NO: 46; and SEQ ID NO: 48) of the variable heavychain region of SEQ ID NO: 42; and the complementarity-determiningregions (SEQ ID NO: 64; SEQ ID NO: 66; and SEQ ID NO: 68) of thevariable light chain region of SEQ ID NO: 62 or sequences that are atleast 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 42; the variable light chainregion of SEQ ID NO: 62; the framework regions (SEQ ID NO: 43; SEQ IDNO: 45; SEQ ID NO: 47; and SEQ ID NO: 49) of the variable heavy chainregion of SEQ ID NO: 42; and the framework regions (SEQ ID NO: 63; SEQID NO: 65; SEQ ID NO: 67; and SEQ ID NO: 69) of the variable light chainregion of SEQ ID NO: 62.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab15, comprising, or alternatively consisting of, SEQ ID NO:41 and SEQ ID NO: 61 or SEQ ID NO: 42 and SEQ ID NO: 62, or an antibodyor antibody fragment comprising the CDRs of Ab15 and having at least oneof the biological activities set forth herein or is an anti-ACTHantibody that competes with Ab15 in binding ACTH, preferably onecontaining sequences that are at least 90%, 95%, 96%, 97%, 98% or 99%identical to that of Ab15 or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab15.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab15, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 42 and the variable lightchain sequence of SEQ ID NO: 62 or sequences that are at least 90%, 95%,96%, 97%, 98% or 99% identical thereto. This embodiment of the inventionfurther includes Fabs containing additions, deletions, and variants ofSEQ ID NO: 42 and/or SEQ ID NO: 62 which retain the binding specificityfor ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) of Ab15.In another embodiment of the invention, anti-ACTH antibodies such asAb15 or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab15 aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab17

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 81) QQQLEESGGGLVKPGGTLTLTCKASGFSFSSGYDICWARQGPGKGLEWIGCIDTGSGNTYYASWAKGRFTISRTSSTTVTLQMTSLTAADTATYFCAKGISSLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 82) QQQLEESGGGLVKPGGTLTLTCKASGFSFSSGYDICWARQGPGKGLEWIGCIDTGSGNTYYASWAKGRFTISRTSSTTVTLQMTSLTAADTATYFCAKGI SSLWGPGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab17 and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 90) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 101) DIVMTQTPASVSEPVGGTVTIKCQASQTISSDLAWYQQKPGQPPKLLIYAASKLTSGVSSRFKGGGTGTQFTLTISDLECADAATYYCQTYYDISDDGCTFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 102) DIVMTQTPASVSEPVGGTVTIKCQASQTISSDLAWYQQKPGQPPKLLIYAASKLTSGVSSRFKGGGTGTQFTLTISDLECADAATYYCQTYYDISDDGCT FGGGTEVVVKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab17 which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 110) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 84; SEQ ID NO: 86; andSEQ ID NO: 88 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 81 or which contain the variable heavy chain sequence of SEQID NO: 82, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 104; SEQ ID NO: 106; and SEQ ID NO:108 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 101or which contain the variable light chain sequence of SEQ ID NO: 102, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; and SEQ ID NO:89 which correspond to the framework regions (FRs or constant regions)of the heavy chain sequence of SEQ ID NO: 81 or the variable heavy chainsequence of SEQ ID NO: 82, and/or one, two, three, or four of thepolypeptide sequences of SEQ ID NO: 103; SEQ ID NO: 105; SEQ ID NO: 107;and SEQ ID NO: 109 which correspond to the framework regions (FRs orconstant regions) of the light chain sequence of SEQ ID NO: 101 or thevariable light chain sequence of SEQ ID NO: 102, or combinations ofthese polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 81 or SEQ ID NO: 82 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 101 or SEQ ID NO: 102 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 84; SEQ ID NO: 86; and SEQ ID NO: 88 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 81 or the variable heavy chainsequence of SEQ ID NO: 82 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 104; SEQ ID NO: 106; and SEQ ID NO: 108 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 101 or the variable light chainsequence of SEQ ID NO: 102 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 83; SEQ ID NO: 85; SEQ ID NO: 87; and SEQ ID NO: 89 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 81 or the variable heavy chainsequence of SEQ ID NO: 82 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 103; SEQ ID NO: 105; SEQ ID NO: 107; and SEQ IDNO: 109 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 101 or the variablelight chain sequence of SEQ ID NO: 102 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 82; the variable lightchain region of SEQ ID NO: 102; the complementarity-determining regions(SEQ ID NO: 84; SEQ ID NO: 86; and SEQ ID NO: 88) of the variable heavychain region of SEQ ID NO: 82; and the complementarity-determiningregions (SEQ ID NO: 104; SEQ ID NO: 106; and SEQ ID NO: 108) of thevariable light chain region of SEQ ID NO: 102 or sequences that are atleast 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 82; the variable light chainregion of SEQ ID NO: 102; the framework regions (SEQ ID NO: 83; SEQ IDNO: 85; SEQ ID NO: 87; and SEQ ID NO: 89) of the variable heavy chainregion of SEQ ID NO: 82; and the framework regions (SEQ ID NO: 103; SEQID NO: 105; SEQ ID NO: 107; and SEQ ID NO: 109) of the variable lightchain region of SEQ ID NO: 102.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab17, comprising, or alternatively consisting of, SEQ ID NO:81 and SEQ ID NO: 101 or SEQ ID NO: 82 and SEQ ID NO: 102, or anantibody or antibody fragment comprising the CDRs of Ab17 and having atleast one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab17 in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab17 or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab17.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab17, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 82 and the variable lightchain sequence of SEQ ID NO: 102 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 82 and/or SEQ ID NO: 102 which retain the bindingspecificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) of Ab17.In another embodiment of the invention, anti-ACTH antibodies such asAb17 or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab17 aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab1.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 121) EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYDMIWVRQAPGKGLESIGMIYDDGDTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 122) EVQLVESGGGLVQPGGSLRLSCAASGFTVSNYDMIWVRQAPGKGLESIGMIYDDGDTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVS NHWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab1.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 130) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 141) DIQMTQSPSTLSASVGDRVTITCQASQSISSYLAWYQQKPGKAPKWYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSGSSYGVGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 142) DIQMTQSPSTLSASVGDRVTITCQASQSISSYLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSGSSYG VGFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab1.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 150) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKEIKVYACEVTHQGLSSPVTKSF NRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 124; SEQ ID NO: 126;and SEQ ID NO: 128 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 121 or which contain the variable heavy chain sequence of SEQID NO: 122, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 144; SEQ ID NO: 146; and SEQ ID NO:148 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 141or which contain the variable light chain sequence of SEQ ID NO: 142, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 123; SEQ ID NO: 125; SEQ ID NO: 127; and SEQ IDNO: 129 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 121 or the variableheavy chain sequence of SEQ ID NO: 122, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 143; SEQ ID NO: 145; SEQ IDNO: 147; and SEQ ID NO: 149 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 141or the variable light chain sequence of SEQ ID NO: 142, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 121 or SEQ ID NO: 122 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 141 or SEQ ID NO: 142 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 124; SEQ ID NO: 126; and SEQ ID NO: 128 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 121 or the variable heavy chainsequence of SEQ ID NO: 122 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 144; SEQ ID NO: 146; and SEQ ID NO: 148 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 141 or the variable light chainsequence of SEQ ID NO: 142 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 123; SEQ ID NO: 125; SEQ ID NO: 127; and SEQ ID NO: 129 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 121 or the variable heavy chainsequence of SEQ ID NO: 122 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 143; SEQ ID NO: 145; SEQ ID NO: 147; and SEQ IDNO: 149 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 141 or the variablelight chain sequence of SEQ ID NO: 142 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 122; the variable lightchain region of SEQ ID NO: 142; the complementarity-determining regions(SEQ ID NO: 124; SEQ ID NO: 126; and SEQ ID NO: 128) of the variableheavy chain region of SEQ ID NO: 122; and thecomplementarity-determining regions (SEQ ID NO: 144; SEQ ID NO: 146; andSEQ ID NO: 148) of the variable light chain region of SEQ ID NO: 142 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 122; the variable light chainregion of SEQ ID NO: 142; the framework regions (SEQ ID NO: 123; SEQ IDNO: 125; SEQ ID NO: 127; and SEQ ID NO: 129) of the variable heavy chainregion of SEQ ID NO: 122; and the framework regions (SEQ ID NO: 143; SEQID NO: 145; SEQ ID NO: 147; and SEQ ID NO: 149) of the variable lightchain region of SEQ ID NO: 142.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab1.H, comprising, or alternatively consisting of, SEQ IDNO: 121 and SEQ ID NO: 141 or SEQ ID NO: 122 and SEQ ID NO: 142, or anantibody or antibody fragment comprising the CDRs of Ab1.H and having atleast one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab1.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab1.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab1.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab1.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 122 and the variable lightchain sequence of SEQ ID NO: 142 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 122 and/or SEQ ID NO: 142 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb1.H. In another embodiment of the invention, anti-ACTH antibodies suchas Ab1.H or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab1.H aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab2.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 161) EVQLVESGGGLVQPGGSLRLSCAASGFTVSKYDMIWVRQAPGKGLESIGIIYDDGDTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 162) EVQLVESGGGLVQPGGSLRLSCAASGFTVSKYDMIWVRQAPGKGLESIGIIYDDGDTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNI WGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab2.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 170) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 181) DIQMTQSPSTLSASVGDRVTITCQASQSISNYLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYEGSSSSSYGVGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 182) DIQMTQSPSTLSASVGDRVTITCQASQSISNYLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYEGSSSSSYGVG FGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab2.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 190) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 164; SEQ ID NO: 166;and SEQ ID NO: 168 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 161 or which contain the variable heavy chain sequence of SEQID NO: 162, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 184; SEQ ID NO: 186; and SEQ ID NO:188 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 181or which contain the variable light chain sequence of SEQ ID NO: 182, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 163; SEQ ID NO: 165; SEQ ID NO: 167; and SEQ IDNO: 169 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 161 or the variableheavy chain sequence of SEQ ID NO: 162, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 183; SEQ ID NO: 185; SEQ IDNO: 187; and SEQ ID NO: 189 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 181or the variable light chain sequence of SEQ ID NO: 182, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 161 or SEQ ID NO: 162 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 181 or SEQ ID NO: 182 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 164; SEQ ID NO: 166; and SEQ ID NO: 168 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 161 or the variable heavy chainsequence of SEQ ID NO: 162 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 184; SEQ ID NO: 186; and SEQ ID NO: 188 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 181 or the variable light chainsequence of SEQ ID NO: 182 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 163; SEQ ID NO: 165; SEQ ID NO: 167; and SEQ ID NO: 169 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 161 or the variable heavy chainsequence of SEQ ID NO: 162 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 183; SEQ ID NO: 185; SEQ ID NO: 187; and SEQ IDNO: 189 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 181 or the variablelight chain sequence of SEQ ID NO: 182 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 162; the variable lightchain region of SEQ ID NO: 182; the complementarity-determining regions(SEQ ID NO: 164; SEQ ID NO: 166; and SEQ ID NO: 168) of the variableheavy chain region of SEQ ID NO: 162; and thecomplementarity-determining regions (SEQ ID NO: 184; SEQ ID NO: 186; andSEQ ID NO: 188) of the variable light chain region of SEQ ID NO: 182 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 162; the variable light chainregion of SEQ ID NO: 182; the framework regions (SEQ ID NO: 163; SEQ IDNO: 165; SEQ ID NO: 167; and SEQ ID NO: 169) of the variable heavy chainregion of SEQ ID NO: 162; and the framework regions (SEQ ID NO: 183; SEQID NO: 185; SEQ ID NO: 187; and SEQ ID NO: 189) of the variable lightchain region of SEQ ID NO: 182.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab2.H, comprising, or alternatively consisting of, SEQ IDNO: 161 and SEQ ID NO: 181 or SEQ ID NO: 162 and SEQ ID NO: 182, or anantibody or antibody fragment comprising the CDRs of Ab2.H and having atleast one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab2.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab2.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab2.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab2.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 162 and the variable lightchain sequence of SEQ ID NO: 182 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 162 and/or SEQ ID NO: 182 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb2.H. In another embodiment of the invention, anti-ACTH antibodies suchas Ab2.H or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab2.H aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab3.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 201) EVQLVESGGGLVQPGGSLRLSCAASGSSLSNFDMIWVRQAPGKGLESIGIIYDFGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 202) EVQLVESGGGLVQPGGSLRLSCAASGSSLSNFDMIWVRQAPGKGLESIGIIYDFGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNI WGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab3.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 210) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 221) DIQMTQSPSTLSASVGDRVTITCQASEDISSNLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYGIGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 222) DIQMTQSPSTLSASVGDRVTITCQASEDISSNLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYGIG FGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab3.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 230) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 204; SEQ ID NO: 206;and SEQ ID NO: 208 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 201 or which contain the variable heavy chain sequence of SEQID NO: 202, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 224; SEQ ID NO: 226; and SEQ ID NO:228 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 221or which contain the variable light chain sequence of SEQ ID NO: 222, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 203; SEQ ID NO: 205; SEQ ID NO: 207; and SEQ IDNO: 209 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 201 or the variableheavy chain sequence of SEQ ID NO: 202, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 223; SEQ ID NO: 225; SEQ IDNO: 227; and SEQ ID NO: 229 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 221or the variable light chain sequence of SEQ ID NO: 222, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 201 or SEQ ID NO: 202 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 221 or SEQ ID NO: 222 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 204; SEQ ID NO: 206; and SEQ ID NO: 208 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 201 or the variable heavy chainsequence of SEQ ID NO: 202 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 224; SEQ ID NO: 226; and SEQ ID NO: 228 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 221 or the variable light chainsequence of SEQ ID NO: 222 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 203; SEQ ID NO: 205; SEQ ID NO: 207; and SEQ ID NO: 209 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 201 or the variable heavy chainsequence of SEQ ID NO: 202 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 223; SEQ ID NO: 225; SEQ ID NO: 227; and SEQ IDNO: 229 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 221 or the variablelight chain sequence of SEQ ID NO: 222 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 202; the variable lightchain region of SEQ ID NO: 222; the complementarity-determining regions(SEQ ID NO: 204; SEQ ID NO: 206; and SEQ ID NO: 208) of the variableheavy chain region of SEQ ID NO: 202; and thecomplementarity-determining regions (SEQ ID NO: 224; SEQ ID NO: 226; andSEQ ID NO: 228) of the variable light chain region of SEQ ID NO: 222 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 202; the variable light chainregion of SEQ ID NO: 222; the framework regions (SEQ ID NO: 203; SEQ IDNO: 205; SEQ ID NO: 207; and SEQ ID NO: 209) of the variable heavy chainregion of SEQ ID NO: 202; and the framework regions (SEQ ID NO: 223; SEQID NO: 225; SEQ ID NO: 227; and SEQ ID NO: 229) of the variable lightchain region of SEQ ID NO: 222.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab3.H, comprising, or alternatively consisting of, SEQ IDNO: 201 and SEQ ID NO: 221 or SEQ ID NO: 202 and SEQ ID NO: 222, or anantibody or antibody fragment comprising the CDRs of Ab3.H and having atleast one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab3.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab3.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab3.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab3.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 202 and the variable lightchain sequence of SEQ ID NO: 222 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 202 and/or SEQ ID NO: 222 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb3.H. In another embodiment of the invention, anti-ACTH antibodies suchas Ab3.H or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab3.H aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab4.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 241) EVQLVESGGGLVQPGGSLRLSCAASGFTVSKHDMIWVRQAPGKGLESIGIIYDDGDTYYANSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 242) EVQLVESGGGLVQPGGSLRLSCAASGFTVSKHDMIWVRQAPGKGLESIGIIYDDGDTYYANSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNI WGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab4.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 250) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 261) DIQMTQSPSTLSASVGDRVTITCRASQSISVYLAWYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYGVGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 262) DIQMTQSPSTLSASVGDRVTITCRASQSISVYLAWYQQKPGKAPKLLIYQASKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYG VGFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab4.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 270) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 244; SEQ ID NO: 246;and SEQ ID NO: 248 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 241 or which contain the variable heavy chain sequence of SEQID NO: 242, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 264; SEQ ID NO: 266; and SEQ ID NO:268 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 261or which contain the variable light chain sequence of SEQ ID NO: 262, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 243; SEQ ID NO: 245; SEQ ID NO: 247; and SEQ IDNO: 249 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 241 or the variableheavy chain sequence of SEQ ID NO: 242, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 263; SEQ ID NO: 265; SEQ IDNO: 267; and SEQ ID NO: 269 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 261or the variable light chain sequence of SEQ ID NO: 262, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 241 or SEQ ID NO: 242 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 261 or SEQ ID NO: 262 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 244; SEQ ID NO: 246; and SEQ ID NO: 248 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 241 or the variable heavy chainsequence of SEQ ID NO: 242 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 264; SEQ ID NO: 266; and SEQ ID NO: 268 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 261 or the variable light chainsequence of SEQ ID NO: 262 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 243; SEQ ID NO: 245; SEQ ID NO: 247; and SEQ ID NO: 249 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 241 or the variable heavy chainsequence of SEQ ID NO: 242 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 263; SEQ ID NO: 265; SEQ ID NO: 267; and SEQ IDNO: 269 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 261 or the variablelight chain sequence of SEQ ID NO: 262 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 242; the variable lightchain region of SEQ ID NO: 262; the complementarity-determining regions(SEQ ID NO: 244; SEQ ID NO: 246; and SEQ ID NO: 248) of the variableheavy chain region of SEQ ID NO: 242; and thecomplementarity-determining regions (SEQ ID NO: 264; SEQ ID NO: 266; andSEQ ID NO: 268) of the variable light chain region of SEQ ID NO: 262 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 242; the variable light chainregion of SEQ ID NO: 262; the framework regions (SEQ ID NO: 243; SEQ IDNO: 245; SEQ ID NO: 247; and SEQ ID NO: 249) of the variable heavy chainregion of SEQ ID NO: 242; and the framework regions (SEQ ID NO: 263; SEQID NO: 265; SEQ ID NO: 267; and SEQ ID NO: 269) of the variable lightchain region of SEQ ID NO: 262.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab4.H, comprising, or alternatively consisting of, SEQ IDNO: 241 and SEQ ID NO: 261 or SEQ ID NO: 242 and SEQ ID NO: 262, or anantibody or antibody fragment comprising the CDRs of Ab4.H and having atleast one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab4.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab4.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab4.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab4.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 242 and the variable lightchain sequence of SEQ ID NO: 262 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 242 and/or SEQ ID NO: 262 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb4.H. In another embodiment of the invention, anti-ACTH antibodies suchas Ab4.H or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab4.H aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab6.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 281) EVQLVESGGGLVQPGGSLRLSCAASGFSLTDYAMSWVRQAPGKGLEWIGIISDSGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPEYGYDEYGDWVSDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 282) EVQLVESGGGLVQPGGSLRLSCAASGFSLTDYAMSWVRQAPGKGLEWIGIISDSGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPEYGYDEYGDWVSDLWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab6.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 290) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 301) GSGTEFTLTISSLQPDDFATYYCQSYYYSSSITYHNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 302) DIQMTQSPSTLSASVGDRVTITCQATQSIGNNLAWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYYYSSSITYH NAFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab6.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 310) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 284; SEQ ID NO: 286;and SEQ ID NO: 288 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 281 or which contain the variable heavy chain sequence of SEQID NO: 282, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 304; SEQ ID NO: 306; and SEQ ID NO:308 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 301or which contain the variable light chain sequence of SEQ ID NO: 302, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 283; SEQ ID NO: 285; SEQ ID NO: 287; and SEQ IDNO: 289 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 281 or the variableheavy chain sequence of SEQ ID NO: 282, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 303; SEQ ID NO: 305; SEQ IDNO: 307; and SEQ ID NO: 309 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 301or the variable light chain sequence of SEQ ID NO: 302, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 281 or SEQ ID NO: 282 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 301 or SEQ ID NO: 302 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 284; SEQ ID NO: 286; and SEQ ID NO: 288 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 281 or the variable heavy chainsequence of SEQ ID NO: 282 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 304; SEQ ID NO: 306; and SEQ ID NO: 308 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 301 or the variable light chainsequence of SEQ ID NO: 302 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 283; SEQ ID NO: 285; SEQ ID NO: 287; and SEQ ID NO: 289 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 281 or the variable heavy chainsequence of SEQ ID NO: 282 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 303; SEQ ID NO: 305; SEQ ID NO: 307; and SEQ IDNO: 309 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 301 or the variablelight chain sequence of SEQ ID NO: 302 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 282; the variable lightchain region of SEQ ID NO: 302; the complementarity-determining regions(SEQ ID NO: 284; SEQ ID NO: 286; and SEQ ID NO: 288) of the variableheavy chain region of SEQ ID NO: 282; and thecomplementarity-determining regions (SEQ ID NO: 304; SEQ ID NO: 306; andSEQ ID NO: 308) of the variable light chain region of SEQ ID NO: 302 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 282; the variable light chainregion of SEQ ID NO: 302; the framework regions (SEQ ID NO: 283; SEQ IDNO: 285; SEQ ID NO: 287; and SEQ ID NO: 289) of the variable heavy chainregion of SEQ ID NO: 282; and the framework regions (SEQ ID NO: 303; SEQID NO: 305; SEQ ID NO: 307; and SEQ ID NO: 309) of the variable lightchain region of SEQ ID NO: 302.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab6.H, comprising, or alternatively consisting of, SEQ IDNO: 281 and SEQ ID NO: 301 or SEQ ID NO: 282 and SEQ ID NO: 302, or anantibody or antibody fragment comprising the CDRs of Ab6.H and having atleast one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab6.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab6.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab6.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab6.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 282 and the variable lightchain sequence of SEQ ID NO: 302 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 282 and/or SEQ ID NO: 302 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb6.H. In another embodiment of the invention, anti-ACTH antibodies suchas Ab6.H or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab6.H aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab7.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 321) EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYAMSWVRQAPGKGLEWIGIISDSGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPEYGYDDYGDWVSDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 322) EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYAMSWVRQAPGKGLEWIGIISDSGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPEYGYDDYGDWVSDLWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab7.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 330) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 341) DIQMTQSPSTLSASVGDRVTITCQASQSISDYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYYYSSSITYRNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 342) DIQMTQSPSTLSASVGDRVTITCQASQSISDYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYYYSSSITYR NAFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab7.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 350) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 324; SEQ ID NO: 326;and SEQ ID NO: 328 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 321 or which contain the variable heavy chain sequence of SEQID NO: 322, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 344; SEQ ID NO: 346; and SEQ ID NO:348 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 341or which contain the variable light chain sequence of SEQ ID NO: 342, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 323; SEQ ID NO: 325; SEQ ID NO: 327; and SEQ IDNO: 329 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 321 or the variableheavy chain sequence of SEQ ID NO: 322, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 343; SEQ ID NO: 345; SEQ IDNO: 347; and SEQ ID NO: 349 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 341or the variable light chain sequence of SEQ ID NO: 342, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 321 or SEQ ID NO: 322 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 341 or SEQ ID NO: 342 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 324; SEQ ID NO: 326; and SEQ ID NO: 328 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 321 or the variable heavy chainsequence of SEQ ID NO: 322 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 344; SEQ ID NO: 346; and SEQ ID NO: 348 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 341 or the variable light chainsequence of SEQ ID NO: 342 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 323; SEQ ID NO: 325; SEQ ID NO: 327; and SEQ ID NO: 329 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 321 or the variable heavy chainsequence of SEQ ID NO: 322 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 343; SEQ ID NO: 345; SEQ ID NO: 347; and SEQ IDNO: 349 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 341 or the variablelight chain sequence of SEQ ID NO: 342 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 322; the variable lightchain region of SEQ ID NO: 342; the complementarity-determining regions(SEQ ID NO: 324; SEQ ID NO: 326; and SEQ ID NO: 328) of the variableheavy chain region of SEQ ID NO: 322; and thecomplementarity-determining regions (SEQ ID NO: 344; SEQ ID NO: 346; andSEQ ID NO: 348) of the variable light chain region of SEQ ID NO: 342 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 322; the variable light chainregion of SEQ ID NO: 342; the framework regions (SEQ ID NO: 323; SEQ IDNO: 325; SEQ ID NO: 327; and SEQ ID NO: 329) of the variable heavy chainregion of SEQ ID NO: 322; and the framework regions (SEQ ID NO: 343; SEQID NO: 345; SEQ ID NO: 347; and SEQ ID NO: 349) of the variable lightchain region of SEQ ID NO: 342.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab7.H, comprising, or alternatively consisting of, SEQ IDNO: 321 and SEQ ID NO: 341 or SEQ ID NO: 322 and SEQ ID NO: 342, or anantibody or antibody fragment comprising the CDRs of Ab7.H and having atleast one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab7.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab7.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab7.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab7.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 322 and the variable lightchain sequence of SEQ ID NO: 342 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 322 and/or SEQ ID NO: 342 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb7.H. In another embodiment of the invention, anti-ACTH antibodies suchas Ab7.H or Fab fragments thereof may be produced via expression inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab7.H aswell as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab7A.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 361) EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYAMSWVRQAPGKGLEWIGIISDSGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPEYGYDDYGDWVSDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 362) EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYAMSWVRQAPGKGLEWIGIISDSGSTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREPEYGYDDYGDWVSDLWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab7A.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 370) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 381) ADIQMTQSPSTLSASVGDRVTITCQASQSISDYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYYYSSSITYRNAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 382) ADIQMTQSPSTLSASVGDRVTITCQASQSISDYLSWYQQKPGKAPKLLIYRASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYYYSSSITY RNAFGGGTKVEIKR .

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab7A.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 390) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 364; SEQ ID NO: 366;and SEQ ID NO: 368 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 361 or which contain the variable heavy chain sequence of SEQID NO: 362, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 384; SEQ ID NO: 386; and SEQ ID NO:388 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 381or which contain the variable light chain sequence of SEQ ID NO: 382, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 363; SEQ ID NO: 365; SEQ ID NO: 367; and SEQ IDNO: 369 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 361 or the variableheavy chain sequence of SEQ ID NO: 362, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 383; SEQ ID NO: 385; SEQ IDNO: 387; and SEQ ID NO: 389 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 381or the variable light chain sequence of SEQ ID NO: 382, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 361 or SEQ ID NO: 362 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 381 or SEQ ID NO: 382 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 364; SEQ ID NO: 366; and SEQ ID NO: 368 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 361 or the variable heavy chainsequence of SEQ ID NO: 362 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 384; SEQ ID NO: 386; and SEQ ID NO: 388 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 381 or the variable light chainsequence of SEQ ID NO: 382 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 363; SEQ ID NO: 365; SEQ ID NO: 367; and SEQ ID NO: 369 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 361 or the variable heavy chainsequence of SEQ ID NO: 362 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 383; SEQ ID NO: 385; SEQ ID NO: 387; and SEQ IDNO: 389 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 381 or the variablelight chain sequence of SEQ ID NO: 382 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 362; the variable lightchain region of SEQ ID NO: 382; the complementarity-determining regions(SEQ ID NO: 364; SEQ ID NO: 366; and SEQ ID NO: 368) of the variableheavy chain region of SEQ ID NO: 362; and thecomplementarity-determining regions (SEQ ID NO: 384; SEQ ID NO: 386; andSEQ ID NO: 388) of the variable light chain region of SEQ ID NO: 382 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 362; the variable light chainregion of SEQ ID NO: 382; the framework regions (SEQ ID NO: 363; SEQ IDNO: 365; SEQ ID NO: 367; and SEQ ID NO: 369) of the variable heavy chainregion of SEQ ID NO: 362; and the framework regions (SEQ ID NO: 383; SEQID NO: 385; SEQ ID NO: 387; and SEQ ID NO: 389) of the variable lightchain region of SEQ ID NO: 382.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab7A.H, comprising, or alternatively consisting of, SEQ IDNO: 361 and SEQ ID NO: 381 or SEQ ID NO: 362 and SEQ ID NO: 382, or anantibody or antibody fragment comprising the CDRs of Ab7A.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab7A.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab7A.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab7A.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab7A.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 362 and the variable lightchain sequence of SEQ ID NO: 382 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 362 and/or SEQ ID NO: 382 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb7A.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab7A.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab7A.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab10.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 401) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSADMIWVRQAPGKGLESIGMIYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSSVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 402) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSADMIWVRQAPGKGLESIGMIYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVS SVWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab10.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 410) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 421) DIQMTQSPSTLSASVGDRVTITCQASENIYRSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYGVGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 422) DIQMTQSPSTLSASVGDRVTITCQASENIYRSLAWYQQKPGKAPKKKIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYG VGFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab10.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 430) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 404; SEQ ID NO: 406;and SEQ ID NO: 408 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 401 or which contain the variable heavy chain sequence of SEQID NO: 402, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 424; SEQ ID NO: 426; and SEQ ID NO:428 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 421or which contain the variable light chain sequence of SEQ ID NO: 422, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 403; SEQ ID NO: 405; SEQ ID NO: 407; and SEQ IDNO: 409 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 401 or the variableheavy chain sequence of SEQ ID NO: 402, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 423; SEQ ID NO: 425; SEQ IDNO: 427; and SEQ ID NO: 429 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 421or the variable light chain sequence of SEQ ID NO: 422, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 401 or SEQ ID NO: 402 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 421 or SEQ ID NO: 422 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 404; SEQ ID NO: 406; and SEQ ID NO: 408 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 401 or the variable heavy chainsequence of SEQ ID NO: 402 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 424; SEQ ID NO: 426; and SEQ ID NO: 428 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 421 or the variable light chainsequence of SEQ ID NO: 422 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 403; SEQ ID NO: 405; SEQ ID NO: 407; and SEQ ID NO: 409 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 401 or the variable heavy chainsequence of SEQ ID NO: 402 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 423; SEQ ID NO: 425; SEQ ID NO: 427; and SEQ IDNO: 429 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 421 or the variablelight chain sequence of SEQ ID NO: 422 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 402; the variable lightchain region of SEQ ID NO: 422; the complementarity-determining regions(SEQ ID NO: 404; SEQ ID NO: 406; and SEQ ID NO: 408) of the variableheavy chain region of SEQ ID NO: 402; and thecomplementarity-determining regions (SEQ ID NO: 424; SEQ ID NO: 426; andSEQ ID NO: 428) of the variable light chain region of SEQ ID NO: 422 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 402; the variable light chainregion of SEQ ID NO: 422; the framework regions (SEQ ID NO: 403; SEQ IDNO: 405; SEQ ID NO: 407; and SEQ ID NO: 409) of the variable heavy chainregion of SEQ ID NO: 402; and the framework regions (SEQ ID NO: 423; SEQID NO: 425; SEQ ID NO: 427; and SEQ ID NO: 429) of the variable lightchain region of SEQ ID NO: 422.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab10.H, comprising, or alternatively consisting of, SEQ IDNO: 401 and SEQ ID NO: 421 or SEQ ID NO: 402 and SEQ ID NO: 422, or anantibody or antibody fragment comprising the CDRs of Ab10.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab10.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab10.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab10.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab10.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 402 and the variable lightchain sequence of SEQ ID NO: 422 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 402 and/or SEQ ID NO: 422 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb10.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab10.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab10.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab11.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 441) EVQLVESGGGLVQPGGSLRLSCAASGFTVSAYDILWVRQAPGKGLESIGMMYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 442) EVQLVESGGGLVQPGGSLRLSCAASGFTVSAYDILWVRQAPGKGLESIGMMYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVS NIWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab11.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 450) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 461) DIQMTQSPSTLSASVGDRVTITCQASQSIDSSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSYYGIGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 462) DIQMTQSPSTLSASVGDRVTITCQASQSIDSSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSYYG IGFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab11.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 470) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 444; SEQ ID NO: 446;and SEQ ID NO: 448 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 441 or which contain the variable heavy chain sequence of SEQID NO: 442, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 464; SEQ ID NO: 466; and SEQ ID NO:468 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 461or which contain the variable light chain sequence of SEQ ID NO: 462, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 443; SEQ ID NO: 445; SEQ ID NO: 447; and SEQ IDNO: 449 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 441 or the variableheavy chain sequence of SEQ ID NO: 442, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 463; SEQ ID NO: 465; SEQ IDNO: 467; and SEQ ID NO: 469 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 461or the variable light chain sequence of SEQ ID NO: 462, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 441 or SEQ ID NO: 442 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 461 or SEQ ID NO: 462 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 444; SEQ ID NO: 446; and SEQ ID NO: 448 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 441 or the variable heavy chainsequence of SEQ ID NO: 442 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 464; SEQ ID NO: 466; and SEQ ID NO: 468 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 461 or the variable light chainsequence of SEQ ID NO: 462 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 443; SEQ ID NO: 445; SEQ ID NO: 447; and SEQ ID NO: 449 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 441 or the variable heavy chainsequence of SEQ ID NO: 442 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 463; SEQ ID NO: 465; SEQ ID NO: 467; and SEQ IDNO: 469 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 461 or the variablelight chain sequence of SEQ ID NO: 462 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 442; the variable lightchain region of SEQ ID NO: 462; the complementarity-determining regions(SEQ ID NO: 444; SEQ ID NO: 446; and SEQ ID NO: 448) of the variableheavy chain region of SEQ ID NO: 442; and thecomplementarity-determining regions (SEQ ID NO: 464; SEQ ID NO: 466; andSEQ ID NO: 468) of the variable light chain region of SEQ ID NO: 462 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 442; the variable light chainregion of SEQ ID NO: 462; the framework regions (SEQ ID NO: 443; SEQ IDNO: 445; SEQ ID NO: 447; and SEQ ID NO: 449) of the variable heavy chainregion of SEQ ID NO: 442; and the framework regions (SEQ ID NO: 463; SEQID NO: 465; SEQ ID NO: 467; and SEQ ID NO: 469) of the variable lightchain region of SEQ ID NO: 462.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab11.H, comprising, or alternatively consisting of, SEQ IDNO: 441 and SEQ ID NO: 461 or SEQ ID NO: 442 and SEQ ID NO: 462, or anantibody or antibody fragment comprising the CDRs of Ab11.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab1.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab11.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab11.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab11.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 442 and the variable lightchain sequence of SEQ ID NO: 462 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 442 and/or SEQ ID NO: 462 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb11.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab11.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab11.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab11A.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 481) EVQLVESGGGLVQPGGSLRLSCAASGFTVSAYDILWVRQAPGKGLESIGMMYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 482) EVQLVESGGGLVQPGGSLRLSCAASGFTVSAYDILWVRQAPGKGLESIGMMYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVS NIWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab11A.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 490) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 501) DIQMTQSPSTLSASVGDRVTITCQASQSIGSSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYEGSSSSYYGIGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 502) DIQMTQSPSTLSASVGDRVTITCQASQSIGSSLAWYQQKPGKAPKLLIYSASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYEGSSSSYYG IGFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab11A.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 510) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 484; SEQ ID NO: 486;and SEQ ID NO: 488 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 481 or which contain the variable heavy chain sequence of SEQID NO: 482, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 504; SEQ ID NO: 506; and SEQ ID NO:508 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 501or which contain the variable light chain sequence of SEQ ID NO: 502, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 483; SEQ ID NO: 485; SEQ ID NO: 487; and SEQ IDNO: 489 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 481 or the variableheavy chain sequence of SEQ ID NO: 482, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 503; SEQ ID NO: 505; SEQ IDNO: 507; and SEQ ID NO: 509 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 501or the variable light chain sequence of SEQ ID NO: 502, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 481 or SEQ ID NO: 482 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 501 or SEQ ID NO: 502 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 484; SEQ ID NO: 486; and SEQ ID NO: 488 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 481 or the variable heavy chainsequence of SEQ ID NO: 482 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 504; SEQ ID NO: 506; and SEQ ID NO: 508 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 501 or the variable light chainsequence of SEQ ID NO: 502 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 483; SEQ ID NO: 485; SEQ ID NO: 487; and SEQ ID NO: 489 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 481 or the variable heavy chainsequence of SEQ ID NO: 482 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 503; SEQ ID NO: 505; SEQ ID NO: 507; and SEQ IDNO: 509 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 501 or the variablelight chain sequence of SEQ ID NO: 502 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 482; the variable lightchain region of SEQ ID NO: 502; the complementarity-determining regions(SEQ ID NO: 484; SEQ ID NO: 486; and SEQ ID NO: 488) of the variableheavy chain region of SEQ ID NO: 482; and thecomplementarity-determining regions (SEQ ID NO: 504; SEQ ID NO: 506; andSEQ ID NO: 508) of the variable light chain region of SEQ ID NO: 502 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 482; the variable light chainregion of SEQ ID NO: 502; the framework regions (SEQ ID NO: 483; SEQ IDNO: 485; SEQ ID NO: 487; and SEQ ID NO: 489) of the variable heavy chainregion of SEQ ID NO: 482; and the framework regions (SEQ ID NO: 503; SEQID NO: 505; SEQ ID NO: 507; and SEQ ID NO: 509) of the variable lightchain region of SEQ ID NO: 502.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab11A.H, comprising, or alternatively consisting of, SEQ IDNO: 481 and SEQ ID NO: 501 or SEQ ID NO: 482 and SEQ ID NO: 502, or anantibody or antibody fragment comprising the CDRs of Ab11A.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab11A.H in binding ACTH,preferably one containing sequences that are at least 90%, 95%, 96%,97%, 98% or 99% identical to that of Ab11A.H or an antibody that bindsto the same or overlapping epitope(s) on ACTH as Ab11A.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab11A.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 482 and the variable lightchain sequence of SEQ ID NO: 502 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 482 and/or SEQ ID NO: 502 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb11A.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab11A.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab11A.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab12.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 521) EVQLVESGGGLVQPGGSLRLSCAASGSSLSDYDMIWVRQAPGKGLESIGIIYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVSNMWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 522) EVQLVESGGGLVQPGGSLRLSCAASGSSLSDYDMIWVRQAPGKGLESIGIIYDDGDTYYATSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVKGVS NMWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab12.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 530) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 541) DIQMTQSPSTLSASVGDRVTITCQASQSIGSSLAWYQQKPGKAPKLLIYAASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYGVGFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 542) DIQMTQSPSTLSASVGDRVTITCQASQSIGSSLAWYQQKPGKAPKLLIYAASTLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQSYDGSSSSSYG VGFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab12.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 550) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 524; SEQ ID NO: 526;and SEQ ID NO: 528 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 521 or which contain the variable heavy chain sequence of SEQID NO: 522, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 544; SEQ ID NO: 546; and SEQ ID NO:548 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 541or which contain the variable light chain sequence of SEQ ID NO: 542, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 523; SEQ ID NO: 525; SEQ ID NO: 527; and SEQ IDNO: 529 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 521 or the variableheavy chain sequence of SEQ ID NO: 522, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 543; SEQ ID NO: 545; SEQ IDNO: 547; and SEQ ID NO: 549 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 541or the variable light chain sequence of SEQ ID NO: 542, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 521 or SEQ ID NO: 522 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 541 or SEQ ID NO: 542 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 524; SEQ ID NO: 526; and SEQ ID NO: 528 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 521 or the variable heavy chainsequence of SEQ ID NO: 522 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 544; SEQ ID NO: 546; and SEQ ID NO: 548 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 541 or the variable light chainsequence of SEQ ID NO: 542 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 523; SEQ ID NO: 525; SEQ ID NO: 527; and SEQ ID NO: 529 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 521 or the variable heavy chainsequence of SEQ ID NO: 522 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 543; SEQ ID NO: 545; SEQ ID NO: 547; and SEQ IDNO: 549 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 541 or the variablelight chain sequence of SEQ ID NO: 542 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 522; the variable lightchain region of SEQ ID NO: 542; the complementarity-determining regions(SEQ ID NO: 524; SEQ ID NO: 526; and SEQ ID NO: 528) of the variableheavy chain region of SEQ ID NO: 522; and thecomplementarity-determining regions (SEQ ID NO: 544; SEQ ID NO: 546; andSEQ ID NO: 548) of the variable light chain region of SEQ ID NO: 542 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 522; the variable light chainregion of SEQ ID NO: 542; the framework regions (SEQ ID NO: 523; SEQ IDNO: 525; SEQ ID NO: 527; and SEQ ID NO: 529) of the variable heavy chainregion of SEQ ID NO: 522; and the framework regions (SEQ ID NO: 543; SEQID NO: 545; SEQ ID NO: 547; and SEQ ID NO: 549) of the variable lightchain region of SEQ ID NO: 542.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab12.H, comprising, or alternatively consisting of, SEQ IDNO: 521 and SEQ ID NO: 541 or SEQ ID NO: 522 and SEQ ID NO: 542, or anantibody or antibody fragment comprising the CDRs of Ab12.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab12.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab12.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab12.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab12.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 522 and the variable lightchain sequence of SEQ ID NO: 542 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 522 and/or SEQ ID NO: 542 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb12.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab12.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab12.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab13.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 561) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAKGISSIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 562) EVQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCA KGISSIWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab13.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 570) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 581) DIQMTQSPSTLSASVGDRVTITCQASQTISSDLAWYQQKPGKAPKLLIYAASKLTSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQTYYDIIDDGATFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 582) DIQMTQSPSTLSASVGDRVTITCQASQTISSDLAWYQQKPGKAPKLLIYAASKLTSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQTYYDIIDDG ATFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab13.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 590) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 564; SEQ ID NO: 566;and SEQ ID NO: 568 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 561 or which contain the variable heavy chain sequence of SEQID NO: 562, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 584; SEQ ID NO: 586; and SEQ ID NO:588 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 581or which contain the variable light chain sequence of SEQ ID NO: 582, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 563; SEQ ID NO: 565; SEQ ID NO: 567; and SEQ IDNO: 569 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 561 or the variableheavy chain sequence of SEQ ID NO: 562, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 583; SEQ ID NO: 585; SEQ IDNO: 587; and SEQ ID NO: 589 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 581or the variable light chain sequence of SEQ ID NO: 582, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 561 or SEQ ID NO: 562 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 581 or SEQ ID NO: 582 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 564; SEQ ID NO: 566; and SEQ ID NO: 568 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 561 or the variable heavy chainsequence of SEQ ID NO: 562 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 584; SEQ ID NO: 586; and SEQ ID NO: 588 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 581 or the variable light chainsequence of SEQ ID NO: 582 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 563; SEQ ID NO: 565; SEQ ID NO: 567; and SEQ ID NO: 569 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 561 or the variable heavy chainsequence of SEQ ID NO: 562 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 583; SEQ ID NO: 585; SEQ ID NO: 587; and SEQ IDNO: 589 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 581 or the variablelight chain sequence of SEQ ID NO: 582 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 562; the variable lightchain region of SEQ ID NO: 582; the complementarity-determining regions(SEQ ID NO: 564; SEQ ID NO: 566; and SEQ ID NO: 568) of the variableheavy chain region of SEQ ID NO: 562; and thecomplementarity-determining regions (SEQ ID NO: 584; SEQ ID NO: 586; andSEQ ID NO: 588) of the variable light chain region of SEQ ID NO: 582 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 562; the variable light chainregion of SEQ ID NO: 582; the framework regions (SEQ ID NO: 563; SEQ IDNO: 565; SEQ ID NO: 567; and SEQ ID NO: 569) of the variable heavy chainregion of SEQ ID NO: 562; and the framework regions (SEQ ID NO: 583; SEQID NO: 585; SEQ ID NO: 587; and SEQ ID NO: 589) of the variable lightchain region of SEQ ID NO: 582.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab13.H, comprising, or alternatively consisting of, SEQ IDNO: 561 and SEQ ID NO: 581 or SEQ ID NO: 562 and SEQ ID NO: 582, or anantibody or antibody fragment comprising the CDRs of Ab13.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab13.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab13.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab13.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab13.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 562 and the variable lightchain sequence of SEQ ID NO: 582 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 562 and/or SEQ ID NO: 582 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb13.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab13.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab13.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab15.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 601) QQQLVESGGGLVQPGGSLRLSCAASGFTVSDTYDMCWVRQAPGKGLEWIGCIDTGSGDTYYPTSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGVSSLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK.

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 602) QQQLVESGGGLVQPGGSLRLSCAASGFTVSDTYDMCWVRQAPGKGLEWIGCIDTGSGDTYYPTSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KGVSSLWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab15.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 610) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 621) DIQMTQSPSTLSASVGDRVTITCQASEDIESDLAWYQQKPGKAPKLLIYGASTLKSGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQTYYDMADDGASFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 622) DIQMTQSPSTLSASVGDRVTITCQASEDIESDLAWYQQKPGKAPKLLIYGASTLKSGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQTYYDMADDG ASFGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab15.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 630) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVT KSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 604; SEQ ID NO: 606;and SEQ ID NO: 608 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 601 or which contain the variable heavy chain sequence of SEQID NO: 602, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 624; SEQ ID NO: 626; and SEQ ID NO:628 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 621or which contain the variable light chain sequence of SEQ ID NO: 622, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 603; SEQ ID NO: 605; SEQ ID NO: 607; and SEQ IDNO: 609 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 601 or the variableheavy chain sequence of SEQ ID NO: 602, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 623; SEQ ID NO: 625; SEQ IDNO: 627; and SEQ ID NO: 629 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 621or the variable light chain sequence of SEQ ID NO: 622, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 601 or SEQ ID NO: 602 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 621 or SEQ ID NO: 622 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 604; SEQ ID NO: 606; and SEQ ID NO: 608 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 601 or the variable heavy chainsequence of SEQ ID NO: 602 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 624; SEQ ID NO: 626; and SEQ ID NO: 628 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 621 or the variable light chainsequence of SEQ ID NO: 622 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 603; SEQ ID NO: 605; SEQ ID NO: 607; and SEQ ID NO: 609 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 601 or the variable heavy chainsequence of SEQ ID NO: 602 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 623; SEQ ID NO: 625; SEQ ID NO: 627; and SEQ IDNO: 629 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 621 or the variablelight chain sequence of SEQ ID NO: 622 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 602; the variable lightchain region of SEQ ID NO: 622; the complementarity-determining regions(SEQ ID NO: 604; SEQ ID NO: 606; and SEQ ID NO: 608) of the variableheavy chain region of SEQ ID NO: 602; and thecomplementarity-determining regions (SEQ ID NO: 624; SEQ ID NO: 626; andSEQ ID NO: 628) of the variable light chain region of SEQ ID NO: 622 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 602; the variable light chainregion of SEQ ID NO: 622; the framework regions (SEQ ID NO: 603; SEQ IDNO: 605; SEQ ID NO: 607; and SEQ ID NO: 609) of the variable heavy chainregion of SEQ ID NO: 602; and the framework regions (SEQ ID NO: 623; SEQID NO: 625; SEQ ID NO: 627; and SEQ ID NO: 629) of the variable lightchain region of SEQ ID NO: 622.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab15.H, comprising, or alternatively consisting of, SEQ IDNO: 601 and SEQ ID NO: 621 or SEQ ID NO: 602 and SEQ ID NO: 622, or anantibody or antibody fragment comprising the CDRs of Ab15.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab15.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab15.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab15.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab15.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 602 and the variable lightchain sequence of SEQ ID NO: 622 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 602 and/or SEQ ID NO: 622 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb15.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab15.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab15.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

Antibody Ab17.H

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess a heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 641) QQQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGISSLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK. 

In one embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variableheavy chain sequence comprising the sequence set forth below:

(SEQ ID NO: 642) QQQLVESGGGLVQPGGSLRLSCAASGFTVSSGYDICWVRQAPGKGLEWIGCIDTGSGNTYYASSAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA KGISSLWGQGTLVTVSS.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that possess the sameepitopic specificity as Ab17.H and which contain a constant heavy chainsequence comprising the sequence set forth below:

(SEQ ID NO: 650) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a light chainsequence comprising the sequence set forth below:

(SEQ ID NO: 661) DIQMTQSPSTLSASVGDRVTITCQASQTISSDLAWYQQKPGKAPKLLIYAASKLTSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQTYYDISDDGATFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain a variablelight chain sequence comprising the sequence set forth below:

(SEQ ID NO: 662) DIQMTQSPSTLSASVGDRVTITCQASQTISSDLAWYQQKPGKAPKLLIYAASKLTSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQTYYDISDDGAT FGGGTKVEIKR.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that bind the same epitopeas Ab17.H which contain a constant light chain sequence comprising thesequence set forth below:

(SEQ ID NO: 670) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

In another embodiment, the invention includes antibodies and antibodyfragments having binding specificity to ACTH that contain one, two, orthree of the polypeptide sequences of SEQ ID NO: 644; SEQ ID NO: 646;and SEQ ID NO: 648 which correspond to the complementarity-determiningregions (CDRs, or hypervariable regions) of the heavy chain sequence ofSEQ ID NO: 641 or which contain the variable heavy chain sequence of SEQID NO: 642, and/or which further contain one, two, or three of thepolypeptide sequences of SEQ ID NO: 664; SEQ ID NO: 666; and SEQ ID NO:668 which correspond to the complementarity-determining regions (CDRs,or hypervariable regions) of the light chain sequence of SEQ ID NO: 661or which contain the variable light chain sequence of SEQ ID NO: 662, orantibodies or fragments containing combinations of sequences which areat least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical thereto. Inanother embodiment of the invention, the antibodies of the invention orfragments thereof comprise, or alternatively consist of, combinations ofone or more of the exemplified variable heavy chain and variable lightchain sequences, or the heavy chain and light chain sequences set forthabove, or sequences that are at least 90% or 95% identical thereto.

The invention further contemplates anti-ACTH antibodies and antibodyfragments comprising one, two, three, or four of the polypeptidesequences of SEQ ID NO: 643; SEQ ID NO: 645; SEQ ID NO: 647; and SEQ IDNO: 649 which correspond to the framework regions (FRs or constantregions) of the heavy chain sequence of SEQ ID NO: 641 or the variableheavy chain sequence of SEQ ID NO: 642, and/or one, two, three, or fourof the polypeptide sequences of SEQ ID NO: 663; SEQ ID NO: 665; SEQ IDNO: 667; and SEQ ID NO: 669 which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 661or the variable light chain sequence of SEQ ID NO: 662, or combinationsof these polypeptide sequences or sequences which are at least 80%, 90%,95%, 96%, 97%, 98% or 99% identical therewith.

In another embodiment of the invention, the antibodies and antibodyfragments of the invention or fragments thereof comprise, oralternatively consist of, combinations of one or more of the FRs, CDRs,the variable heavy chain and variable light chain sequences, and theheavy chain and light chain sequences set forth above, including all ofthem or sequences which are at least 90% or 95% identical thereto.

In another embodiment of the invention, the anti-ACTH antibody orantibody fragments of the invention comprise, or alternatively consistof, the polypeptide sequence of SEQ ID NO: 641 or SEQ ID NO: 642 orpolypeptides that are at least 90% or 95% identical thereto. In anotherembodiment of the invention, the antibody or antibody fragments of theinvention comprise, or alternatively consist of, the polypeptidesequence of SEQ ID NO: 661 or SEQ ID NO: 662 or polypeptides that are atleast 90% or 95% identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 644; SEQ ID NO: 646; and SEQ ID NO: 648 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe heavy chain sequence of SEQ ID NO: 641 or the variable heavy chainsequence of SEQ ID NO: 642 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, or three of the polypeptide sequences of SEQ IDNO: 664; SEQ ID NO: 666; and SEQ ID NO: 668 which correspond to thecomplementarity-determining regions (CDRs, or hypervariable regions) ofthe light chain sequence of SEQ ID NO: 661 or the variable light chainsequence of SEQ ID NO: 662 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the antibody or antibodyfragment having binding specificity to ACTH comprises, or alternativelyconsists of, one, two, three, or four of the polypeptide sequences ofSEQ ID NO: 643; SEQ ID NO: 645; SEQ ID NO: 647; and SEQ ID NO: 649 whichcorrespond to the framework regions (FRs or constant regions) of theheavy chain sequence of SEQ ID NO: 641 or the variable heavy chainsequence of SEQ ID NO: 642 or sequences that are at least 90% or 95%identical thereto.

In a further embodiment of the invention, the subject antibody orantibody fragment having binding specificity to ACTH comprises, oralternatively consists of, one, two, three, or four of the polypeptidesequences of SEQ ID NO: 663; SEQ ID NO: 665; SEQ ID NO: 667; and SEQ IDNO: 669 which correspond to the framework regions (FRs or constantregions) of the light chain sequence of SEQ ID NO: 661 or the variablelight chain sequence of SEQ ID NO: 662 or sequences that are at least90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, antibody or antibody fragments havingbinding specificity to ACTH comprise, or alternatively consist of, one,two, three or more, including all of the following antibody fragments:the variable heavy chain region of SEQ ID NO: 642; the variable lightchain region of SEQ ID NO: 662; the complementarity-determining regions(SEQ ID NO: 644; SEQ ID NO: 646; and SEQ ID NO: 648) of the variableheavy chain region of SEQ ID NO: 642; and thecomplementarity-determining regions (SEQ ID NO: 664; SEQ ID NO: 666; andSEQ ID NO: 668) of the variable light chain region of SEQ ID NO: 662 orsequences that are at least 90% or 95% identical thereto.

The invention also contemplates antibody or antibody fragments thatinclude one or more of the antibody fragments described herein. In oneembodiment of the invention, fragments of the antibodies having bindingspecificity to ACTH comprise, or alternatively consist of, one, two,three or more, including all of the following antibody fragments: thevariable heavy chain region of SEQ ID NO: 642; the variable light chainregion of SEQ ID NO: 662; the framework regions (SEQ ID NO: 643; SEQ IDNO: 645; SEQ ID NO: 647; and SEQ ID NO: 649) of the variable heavy chainregion of SEQ ID NO: 642; and the framework regions (SEQ ID NO: 663; SEQID NO: 665; SEQ ID NO: 667; and SEQ ID NO: 669) of the variable lightchain region of SEQ ID NO: 662.

In a particularly preferred embodiment of the invention, the anti-ACTHantibody is Ab17.H, comprising, or alternatively consisting of, SEQ IDNO: 641 and SEQ ID NO: 661 or SEQ ID NO: 642 and SEQ ID NO: 662, or anantibody or antibody fragment comprising the CDRs of Ab17.H and havingat least one of the biological activities set forth herein or is ananti-ACTH antibody that competes with Ab17.H in binding ACTH, preferablyone containing sequences that are at least 90%, 95%, 96%, 97%, 98% or99% identical to that of Ab17.H or an antibody that binds to the same oroverlapping epitope(s) on ACTH as Ab17.H.

In a further particularly preferred embodiment of the invention,antibody fragments comprise, or alternatively consist of, Fab (fragmentantigen binding) fragments having binding specificity for ACTH. Withrespect to antibody Ab17.H, the Fab fragment preferably includes thevariable heavy chain sequence of SEQ ID NO: 642 and the variable lightchain sequence of SEQ ID NO: 662 or sequences that are at least 90%,95%, 96%, 97%, 98% or 99% identical thereto. This embodiment of theinvention further includes Fabs containing additions, deletions, andvariants of SEQ ID NO: 642 and/or SEQ ID NO: 662 which retain thebinding specificity for ACTH.

In one embodiment of the invention described herein (infra), Fabfragments may be produced by enzymatic digestion (e.g., papain) ofAb17.H. In another embodiment of the invention, anti-ACTH antibodiessuch as Ab17.H or Fab fragments thereof may be produced via expressionin mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example haploid or diploidyeast such as haploid or diploid Pichia) and other yeast strains.Suitable Pichia species include, but are not limited to, Pichiapastoris.

In an additional embodiment, the invention is further directed topolynucleotides encoding antibody polypeptides having bindingspecificity to ACTH, including the heavy and/or light chains of Ab17.Has well as fragments, variants, combinations of one or more of the FRs,CDRs, the variable heavy chain and variable light chain sequences, andthe heavy chain and light chain sequences set forth above, including allof them or sequences which are at least 90% or 95% identical thereto.

In another embodiment, the invention contemplates an isolated anti-ACTHantibody comprising a V_(H) polypeptide sequence selected from: SEQ IDNO:2, SEQ ID NO:42, SEQ ID NO:82, SEQ ID NO:122, SEQ ID NO:162, SEQ IDNO:202, SEQ ID NO:242, SEQ ID NO:282, SEQ ID NO:322, SEQ ID NO:362, SEQID NO:402, SEQ ID NO:442, SEQ ID NO:482, SEQ ID NO:522, SEQ ID NO:562,SEQ ID NO:602, SEQ ID NO:642, or a variant thereof; and furthercomprising a V_(L) polypeptide sequence selected from: SEQ ID NO:22, SEQID NO:62, SEQ ID NO: 102, SEQ ID NO: 142, SEQ ID NO:182, SEQ ID NO:222,SEQ ID NO:262, SEQ ID NO:302, SEQ ID NO:342, SEQ ID NO:382, SEQ IDNO:422, SEQ ID NO:462, SEQ ID NO:502, SEQ ID NO:542, SEQ ID NO:582, SEQID NO:622, SEQ ID NO:662, or a variant thereof, wherein one or more ofthe framework residues (FR residues) and/or CDR residues in said V_(H)or V_(L) polypeptide has been substituted with another amino acidresidue resulting in an anti-ACTH antibody that specifically binds ACTH.The invention also includes humanized and chimeric forms of theseantibodies. The chimeric and humanized antibodies may include an Fcderived from IgG1, IgG2, IgG3, or IgG4 constant regions.

In one embodiment of the invention, the chimeric or humanized antibodiesor fragments or V_(H) or V_(L) polypeptides originate or are derivedfrom one or more rabbit antibodies, e.g., a rabbit antibody isolatedfrom a clonal rabbit B cell population.

In some aspects, the invention provides a vector comprising a nucleicacid molecule encoding an anti-ACTH antibody or fragment thereof asdisclosed herein. In some embodiments, the invention provides a hostcell comprising a nucleic acid molecule encoding an anti-ACTH antibodyor fragment thereof as disclosed herein.

In some aspects, the invention provides an isolated antibody or antibodyfragment that competes for binding to ACTH with an antibody or antibodyfragment disclosed herein.

In some aspects, the invention provides a nucleic acid molecule encodingan antibody or antibody fragment as disclosed herein.

In some aspects, the invention provides a pharmaceutical or diagnosticcomposition comprising at least one antibody or antibody fragment asdisclosed herein.

In some aspects, the invention provides a method for treating orpreventing a condition associated with elevated plasma cortisol,corticosterone, and/or aldosterone levels in a subject, comprisingadministering to a subject in need thereof an effective amount of atleast one isolated antibody or antibody fragment as disclosed herein.

In some aspects, the invention provides a method of inhibiting bindingof ACTH to MCR (e.g., MC2R) in a subject comprising administering aneffective amount of at least one antibody or antibody fragment asdisclosed herein.

In some aspects, the invention provides an antibody or antibody fragmentthat selectively binds to ACTH, wherein the antibody or antibodyfragment binds to ACTH with a K_(D) of less than or equal to 5×10⁻⁵ M,10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, or 10⁻¹³ M; preferably, with a K_(D) of less than or equal to5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, or 10⁻¹² M; morepreferably, with a K_(D) that is less than about 100 nM, less than about50 pM, less than about 40 pM, less than about 25 pM, less than about 1pM, between about 10 pM and about 100 pM, between about 1 pM and about100 pM, or between about 1 pM and about 10 pM.

The inventive antibodies and fragments thereof may be modifiedpost-translationally to add effector moieties such as chemical linkers,detectable moieties such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent moieties, or functional moieties such as for examplestreptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, andradioactive materials.

Antibodies or fragments thereof may also be chemically modified toprovide additional advantages such as increased solubility, stabilityand circulating time (in vivo half-life) of the polypeptide, ordecreased immunogenicity (See U.S. Pat. No. 4,179,337). The chemicalmoieties for derivatization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The antibodies and fragments thereof may be modified at randompositions within the molecule, or at predetermined positions within themolecule and may include one, two, three or more attached chemicalmoieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa. Branchedpolyethylene glycols are described, for example, in U.S. Pat. No.5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996);Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); andCaliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures ofeach of which are incorporated herein by reference.

There are a number of attachment methods available to those skilled inthe art, See e.g., EP 0 401 384, herein incorporated by reference(coupling PEG to G-CSF), See also Malik et al., Exp. Hematol.20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresylchloride). For example, polyethylene glycol may be covalently boundthrough amino acid residues via a reactive group, such as, a free aminoor carboxyl group. Reactive groups are those to which an activatedpolyethylene glycol molecule may be bound. The amino acid residueshaving a free amino group may include lysine residues and the N-terminalamino acid residues; those having a free carboxyl group may includeaspartic acid residues glutamic acid residues and the C-terminal aminoacid residue. Sulfhydryl groups may also be used as a reactive group forattaching the polyethylene glycol molecules. Preferred for therapeuticpurposes is attachment at an amino group, such as attachment at theN-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to polypeptides via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) or to more than one type ofamino acid residue (e.g., lysine, histidine, aspartic acid, glutamicacid, cysteine and combinations thereof).

Alternatively, antibodies or fragments thereof may have increased invivo half-lives via fusion with albumin (including but not limited torecombinant human serum albumin or fragments or variants thereof (See,e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622,and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporatedby reference in their entirety)) or other circulating blood proteinssuch as transferrin or ferritin. In a preferred embodiment, polypeptidesand/or antibodies of the present invention (including fragments orvariants thereof) are fused with the mature form of human serum albumin(i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and2 of EP Patent 0 322 094) which is herein incorporated by reference inits entirety. Polynucleotides encoding fusion proteins of the inventionare also encompassed by the invention.

Regarding detectable moieties, further exemplary enzymes include, butare not limited to, horseradish peroxidase, acetylcholinesterase,alkaline phosphatase, beta-galactosidase and luciferase. Furtherexemplary fluorescent materials include, but are not limited to,rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone,dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Furtherexemplary chemiluminescent moieties include, but are not limited to,luminol. Further exemplary bioluminescent materials include, but are notlimited to, luciferin and aequorin. Further exemplary radioactivematerials include, but are not limited to, Iodine 125 (¹²⁵I), Carbon 14(¹⁴C), Sulfur 35 (³⁵S), Tritium (³H) and Phosphorus 32 (³²P).

Regarding functional moieties, exemplary cytotoxic agents include, butare not limited to, methotrexate, aminopterin, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine; alkylating agentssuch as mechlorethamine, thioepa chlorambucil, melphalan, carmustine(BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea,cyclothosphamide, mechlorethamine, busulfan, dibromomannitol,streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP)cisplatin and carboplatin (paraplatin); anthracyclines includedaunorubicin (formerly daunomycin), doxorubicin (adriamycin),detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone andbisantrene; antibiotics include dactinomycin (actinomycin D), bleomycin,calicheamicin, mithramycin, and anthramycin (AMC); and antimitoticagents such as the vinca alkaloids, vincristine and vinblastine. Othercytotoxic agents include paclitaxel (Taxol), ricin, pseudomonasexotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide,emetine, etoposide, teniposide, colchicine, dihydroxy anthracin dione,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, puromycin, procarbazine, hydroxyurea, asparaginase,corticosteroids, mitotane (O,P′-(DDD)), interferons, and mixtures ofthese cytotoxic agents.

Further cytotoxic agents include, but are not limited to,chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C,actinomycin D, cyclophosphamide, vincristine and bleomycin. Toxicenzymes from plants and bacteria such as ricin, diphtheria toxin andPseudomonas toxin may be conjugated to the humanized or chimericantibodies, or binding fragments thereof (Youle, et al., PNAS USA77:5483 (1980); Gilliland, et al., PNAS USA 77:4539 (1980); Krolick, etal., PNAS USA 77:5419 (1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described byGoldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention alsorelate to radioimmunoconjugates where a radionuclide that emits alpha orbeta particles is stably coupled to the antibody, or binding fragmentsthereof, with or without the use of a complex-forming agent. Suchradionuclides include beta-emitters such as Phosphorus-32 (³²P),Scandium-47 (⁴⁷Sc), Copper-67 (⁶⁷Cu), Gallium-67 (⁶⁷Ga), Yttrium-88(⁸⁸Y), Yttrium-90 (⁹⁰Y), Iodine-125 (¹²⁵I), Iodine-131 (¹³¹I),Samarium-153 (¹⁵³Sm), Lutetium-177 (¹⁷⁷Lu), Rhenium-186 (¹⁸⁶Re) orRhenium-188 (¹⁸⁸Re), and alpha-emitters such as Astatine-211 (²¹¹At),Lead-212 (²¹²Pb), Bismuth-212 (²¹²Bi) or -213 (²¹³Bi) or Actinium-225(²²⁵Ac).

Methods are known in the art for conjugating an antibody or bindingfragment thereof to a detectable moiety and the like, such as forexample those methods described by Hunter et al, Nature 144:945 (1962);David et al, Biochemistry 13:1014 (1974); Pain et al, J. Immunol. Meth.40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407 (1982).

Embodiments described herein further include variants and equivalentsthat are substantially homologous to the antibodies, antibody fragments,diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variableregions and CDRs set forth herein. These may contain, e.g., conservativesubstitution mutations, (i.e., the substitution of one or more aminoacids by similar amino acids). For example, conservative substitutionrefers to the substitution of an amino acid with another within the samegeneral class, e.g., one acidic amino acid with another acidic aminoacid, one basic amino acid with another basic amino acid, or one neutralamino acid by another neutral amino acid. What is intended by aconservative amino acid substitution is well known in the art.

In another embodiment, the invention contemplates polypeptide sequenceshaving at least 90% or greater sequence homology to any one or more ofthe polypeptide sequences of antibody fragments, variable regions andCDRs set forth herein. More preferably, the invention contemplatespolypeptide sequences having at least 95% or greater sequence homology,even more preferably at least 98% or greater sequence homology, andstill more preferably at least 99% or greater sequence homology to anyone or more of the polypeptide sequences of antibody fragments, variableregions and CDRs set forth herein. Methods for determining homologybetween nucleic acid and amino acid sequences are well known to those ofordinary skill in the art.

In another embodiment, the invention further contemplates theabove-recited polypeptide homologs of the antibody fragments, variableregions and CDRs set forth herein further having anti-ACTH activity.Non-limiting examples of anti-ACTH activity are set forth herein.

In another embodiment, the invention further contemplates the generationand use of antibodies that bind any of the foregoing sequences,including, but not limited to, anti-idiotypic antibodies. In anexemplary embodiment, such an anti-idiotypic antibody could beadministered to a subject who has received an anti-ACTH antibody tomodulate, reduce, or neutralize, the effect of the anti-ACTH antibody.Such antibodies could also be useful for treatment of an autoimmunedisease characterized by the presence of anti-ACTH antibodies. A furtherexemplary use of such antibodies, e.g., anti-idiotypic antibodies, isfor detection of the anti-ACTH antibodies of the present invention, forexample to monitor the levels of the anti-ACTH antibodies present in asubject's blood or other bodily fluids. For example, in one embodiment,the invention provides a method of using the anti-idiotypic antibody tomonitor the in vivo levels of said anti-ACTH antibody or antibodyfragment in a subject or to neutralize said anti-ACTH antibody in asubject being administered said anti-ACTH antibody or antibody fragment.

The present invention also contemplates anti-ACTH antibodies comprisingany of the polypeptide or polynucleotide sequences described hereinsubstituted for any of the other polynucleotide sequences describedherein. For example, without limitation thereto, the present inventioncontemplates antibodies comprising the combination of any of thevariable light chain and variable heavy chain sequences describedherein, and further contemplates antibodies resulting from substitutionof any of the CDR sequences described herein for any of the other CDRsequences described herein.

Exemplary Polynucleotides Encoding Anti-ACTH Antibody Polypeptides

The invention is further directed to polynucleotides encoding antibodypolypeptides having binding specificity to ACTH.

Antibody Ab13

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 1:

(SEQ ID NO: 11) cagcagctggaggagtccgggggaggcctggtcaagcctggaggaaccctgacactcacctgcacagcctctggattctccttcagtagcggctacgacatctgctgggcccgccagggtccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgcgagctgggcgaaaggccgattcaccatgtccagaacctcgtcgaccacggtgactctgcaagtgaccagtctgacagccgcggacacggccacctatttctgtgcgaagggtatttctagtatatggggcccgggcaccctggtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 2:

(SEQ ID NO: 12) cagcagctggaggagtccgggggaggcctggtcaagcctggaggaaccctgacactcacctgcacagcctctggattctccttcagtagcggctacgacatctgctgggcccgccagggtccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgcgagctgggcgaaaggccgattcaccatgtccagaacctcgtcgaccacggtgactctgcaagtgaccagtctgacagccgcggacacggccacctatttctgtgcgaagggtatttctagtatatggggcccgggcaccctggtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 10:

(SEQ ID NO: 20) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO: 21:

(SEQ ID NO: 31) gatattgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgctgcatccaagctgacatctggggtctcatcgcgcttcaaaggcggtggaactgggacacagttcactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaaacatattatgatattattgatgatggttgtactttcggcggagggaccgaggtggtggtcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtg t.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 22:

(SEQ ID NO: 32) gatattgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgctgcatccaagctgacatctggggtctcatcgcgcttcaaaggcggtggaactgggacacagttcactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaaacatattatgatattattgatgatggttgtactttcggcggagggaccgaggtggtggtcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 30:

(SEQ ID NO: 40) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 14; SEQ ID NO: 16; and SEQ ID NO: 18, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 1 orthe variable heavy chain sequence of SEQ ID NO: 2, and/or one or more ofthe polynucleotide sequences of SEQ ID NO: 34; SEQ ID NO: 36 and SEQ IDNO: 38, which correspond to the complementarity-determining regions(CDRs or hypervariable regions) of the light chain sequence of SEQ IDNO: 21 or the variable light chain sequence of SEQ ID NO: 22, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; and SEQ ID NO: 19, whichcorrespond to polynucleotides encoding the framework regions (FRs orconstant regions) of the heavy chain sequence of SEQ ID NO: 1 or thevariable heavy chain sequence of SEQ ID NO: 2, and/or one or more of thepolynucleotide sequences of SEQ ID NO: 33; SEQ ID NO: 35; SEQ ID NO: 37;and SEQ ID NO: 39, which correspond to the framework regions (FRs orconstant regions) of the light chain sequence of SEQ ID NO: 21 or thevariable light chain sequence of SEQ ID NO: 22, or combinations of thesepolynucleotide sequences. In another embodiment of the invention, thepolynucleotides encoding the antibodies of the invention or fragmentsthereof comprise, or alternatively consist of, combinations of one ormore of the FRs, the variable heavy chain and variable light chainsequences, and the heavy chain and light chain sequences set forthabove, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 11 encoding the heavy chain sequence of SEQ IDNO: 1; the polynucleotide SEQ ID NO: 12 encoding the variable heavychain sequence of SEQ ID NO: 2; the polynucleotide SEQ ID NO: 31encoding the light chain sequence of SEQ ID NO: 21; the polynucleotideSEQ ID NO: 32 encoding the variable light chain sequence of SEQ ID NO:22; polynucleotides encoding the complementarity-determining regions(SEQ ID NO: 14; SEQ ID NO: 16; and SEQ ID NO: 18) of the heavy chainsequence of SEQ ID NO: 1 or the variable heavy chain sequence of SEQ IDNO: 2; polynucleotides encoding the complementarity-determining regions(SEQ ID NO: 34; SEQ ID NO: 36; and SEQ ID NO: 38) of the light chainsequence of SEQ ID NO: 21 or the variable light chain sequence of SEQ IDNO: 22; polynucleotides encoding the framework regions (SEQ ID NO: 13;SEQ ID NO: 15; SEQ ID NO: 17; and SEQ ID NO: 19) of the heavy chainsequence of SEQ ID NO: 1 or the variable heavy chain sequence of SEQ IDNO: 2; and polynucleotides encoding the framework regions (SEQ ID NO:33; SEQ ID NO: 35; SEQ ID NO: 37; and SEQ ID NO: 39) of the light chainsequence of SEQ ID NO: 21 or the variable light chain sequence of SEQ IDNO: 22.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab13, the polynucleotidesencoding the full length Ab13 antibody comprise, or alternativelyconsist of, the polynucleotide SEQ ID NO: 11 encoding the heavy chainsequence of SEQ ID NO: 1 and the polynucleotide SEQ ID NO: 31 encodingthe light chain sequence of SEQ ID NO: 21.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab13 following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab13 or Fab fragmentsthereof may be produced via expression of Ab13 polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab15

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 41:

(SEQ ID NO: 51) cagcagcaactggaagagtccgggggaggcctagtcaagcctggaggaaccctgacactcacctgtaaaggctctggaattgccttcagtgacacctacgacatgtgctgggtccgccaggctccggggaaggggctggaatggatcggatgcatcgatactggtagtggtgacacttactacccgacctgggcgaaaggccgattcaccatctccaaaccctcgtcgaccacggtggatctgaaaatgaccagtctgacagccgcggacacggccacatatttctgtgcgaagggtgtttccagtttatggggccaggggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 42:

(SEQ ID NO: 52) cagcagcaactggaagagtccgggggaggcctagtcaagcctggaggaaccctgacactcacctgtaaaggctctggaattgccttcagtgacacctacgacatgtgctgggtccgccaggctccggggaaggggctggaatggatcggatgcatcgatactggtagtggtgacacttactacccgacctgggcgaaaggccgattcaccatctccaaaccctcgtcgaccacggtggatctgaaaatgaccagtctgacagccgcggacacggccacatatttctgtgcgaagggtgtttccagtttatggggccaggggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 50:

(SEQ ID NO: 60) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO: 61:

(SEQ ID NO: 71) gacatcgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtgaggacattgaaagcgatttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatggtgcatccactctgaagtctggggtctcatcaaggttcagaggcagtggatctgggacagagtacactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaaacctattatgatatggctgatgatggttgtagtttcggcggagggaccgaggtggtggtcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtg t.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 62:

(SEQ ID NO: 72) gacatcgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtgaggacattgaaagcgatttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatggtgcatccactctgaagtctggggtctcatcaaggttcagaggcagtggatctgggacagagtacactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaaacctattatgatatggctgatgatggttgtagtttcggcggagggaccgaggtggtggtcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 70:

(SEQ ID NO: 80) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 54; SEQ ID NO: 56; and SEQ ID NO: 58, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 41or the variable heavy chain sequence of SEQ ID NO: 42, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 74; SEQ ID NO: 76 andSEQ ID NO: 78, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 61 or the variable light chain sequence of SEQ ID NO: 62, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 53; SEQ ID NO: 55; SEQ ID NO: 57; and SEQ ID NO: 59, whichcorrespond to polynucleotides encoding the framework regions (FRs orconstant regions) of the heavy chain sequence of SEQ ID NO: 41 or thevariable heavy chain sequence of SEQ ID NO: 42, and/or one or more ofthe polynucleotide sequences of SEQ ID NO: 73; SEQ ID NO: 75; SEQ ID NO:77; and SEQ ID NO: 79, which correspond to the framework regions (FRs orconstant regions) of the light chain sequence of SEQ ID NO: 61 or thevariable light chain sequence of SEQ ID NO: 62, or combinations of thesepolynucleotide sequences. In another embodiment of the invention, thepolynucleotides encoding the antibodies of the invention or fragmentsthereof comprise, or alternatively consist of, combinations of one ormore of the FRs, the variable heavy chain and variable light chainsequences, and the heavy chain and light chain sequences set forthabove, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 51 encoding the heavy chain sequence of SEQ IDNO: 41; the polynucleotide SEQ ID NO: 52 encoding the variable heavychain sequence of SEQ ID NO: 42; the polynucleotide SEQ ID NO: 71encoding the light chain sequence of SEQ ID NO: 61; the polynucleotideSEQ ID NO: 72 encoding the variable light chain sequence of SEQ ID NO:62; polynucleotides encoding the complementarity-determining regions(SEQ ID NO: 54; SEQ ID NO: 56; and SEQ ID NO: 58) of the heavy chainsequence of SEQ ID NO: 41 or the variable heavy chain sequence of SEQ IDNO: 42; polynucleotides encoding the complementarity-determining regions(SEQ ID NO: 74; SEQ ID NO: 76; and SEQ ID NO: 78) of the light chainsequence of SEQ ID NO: 61 or the variable light chain sequence of SEQ IDNO: 62; polynucleotides encoding the framework regions (SEQ ID NO: 53;SEQ ID NO: 55; SEQ ID NO: 57; and SEQ ID NO: 59) of the heavy chainsequence of SEQ ID NO: 41 or the variable heavy chain sequence of SEQ IDNO: 42; and polynucleotides encoding the framework regions (SEQ ID NO:73; SEQ ID NO: 75; SEQ ID NO: 77; and SEQ ID NO: 79) of the light chainsequence of SEQ ID NO: 61 or the variable light chain sequence of SEQ IDNO: 62.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab15, the polynucleotidesencoding the full length Ab15 antibody comprise, or alternativelyconsist of, the polynucleotide SEQ ID NO: 51 encoding the heavy chainsequence of SEQ ID NO: 41 and the polynucleotide SEQ ID NO: 71 encodingthe light chain sequence of SEQ ID NO: 61.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab15 following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab15 or Fab fragmentsthereof may be produced via expression of Ab15 polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab17

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 81:

(SEQ ID NO: 91) cagcagcagctggaggagtccgggggaggcctggtcaagcctggaggaaccctgacactcacctgcaaagcctctggattctccttcagtagcggctacgacatctgctgggcccgccagggtccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgcgagctgggcgaaaggccgattcaccatctccagaacctcgtcgaccacggtgactctgcaaatgaccagtctgacagccgcggacacggccacctatttctgtgcgaagggtatttctagtttatggggcccgggcaccctggtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 82:

(SEQ ID NO: 92) cagcagcagctggaggagtccgggggaggcctggtcaagcctggaggaaccctgacactcacctgcaaagcctctggattctccttcagtagcggctacgacatctgctgggcccgccagggtccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgcgagctgggcgaaaggccgattcaccatctccagaacctcgtcgaccacggtgactctgcaaatgaccagtctgacagccgcggacacggccacctatttctgtgcgaagggtatttctagtttatggggcccgggcaccctggtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 90:

(SEQ ID NO: 100) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:101:

(SEQ ID NO: 111) gatattgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgctgcatccaaactgacatctggggtctcatcgcggttcaaaggcggtggaactgggacacagttcactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaaacctattatgatattagtgatgatggttgtactttcggcggagggaccgaggtggtggtcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtg t.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 102:

(SEQ ID NO: 112) gatattgtgatgacccagactccagcctccgtgtctgaacctgtgggaggcacagtcaccatcaagtgccaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgctgcatccaaactgacatctggggtctcatcgcggttcaaaggcggtggaactgggacacagttcactctcaccatcagcgacctggagtgtgccgatgctgccacttactactgtcaaacctattatgatattagtgatgatggttgtactttcggcggagggaccgaggtggtggtcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 110:

(SEQ ID NO: 120) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 94; SEQ ID NO: 96; and SEQ ID NO: 98, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 81or the variable heavy chain sequence of SEQ ID NO: 82, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 114; SEQ ID NO: 116and SEQ ID NO: 118, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 101 or the variable light chain sequence of SEQ ID NO: 102,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 93; SEQ ID NO: 95; SEQ ID NO: 97; and SEQ ID NO: 99, whichcorrespond to polynucleotides encoding the framework regions (FRs orconstant regions) of the heavy chain sequence of SEQ ID NO: 81 or thevariable heavy chain sequence of SEQ ID NO: 82, and/or one or more ofthe polynucleotide sequences of SEQ ID NO: 113; SEQ ID NO: 115; SEQ IDNO: 117; and SEQ ID NO: 119, which correspond to the framework regions(FRs or constant regions) of the light chain sequence of SEQ ID NO: 101or the variable light chain sequence of SEQ ID NO: 102, or combinationsof these polynucleotide sequences. In another embodiment of theinvention, the polynucleotides encoding the antibodies of the inventionor fragments thereof comprise, or alternatively consist of, combinationsof one or more of the FRs, the variable heavy chain and variable lightchain sequences, and the heavy chain and light chain sequences set forthabove, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 91 encoding the heavy chain sequence of SEQ IDNO: 81; the polynucleotide SEQ ID NO: 92 encoding the variable heavychain sequence of SEQ ID NO: 82; the polynucleotide SEQ ID NO: 111encoding the light chain sequence of SEQ ID NO: 101; the polynucleotideSEQ ID NO: 112 encoding the variable light chain sequence of SEQ ID NO:102; polynucleotides encoding the complementarity-determining regions(SEQ ID NO: 94; SEQ ID NO: 96; and SEQ ID NO: 98) of the heavy chainsequence of SEQ ID NO: 81 or the variable heavy chain sequence of SEQ IDNO: 82; polynucleotides encoding the complementarity-determining regions(SEQ ID NO: 114; SEQ ID NO: 116; and SEQ ID NO: 118) of the light chainsequence of SEQ ID NO: 101 or the variable light chain sequence of SEQID NO: 102; polynucleotides encoding the framework regions (SEQ ID NO:93; SEQ ID NO: 95; SEQ ID NO: 97; and SEQ ID NO: 99) of the heavy chainsequence of SEQ ID NO: 81 or the variable heavy chain sequence of SEQ IDNO: 82; and polynucleotides encoding the framework regions (SEQ ID NO:113; SEQ ID NO: 115; SEQ ID NO: 117; and SEQ ID NO: 119) of the lightchain sequence of SEQ ID NO: 101 or the variable light chain sequence ofSEQ ID NO: 102.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab17, the polynucleotidesencoding the full length Ab17 antibody comprise, or alternativelyconsist of, the polynucleotide SEQ ID NO: 91 encoding the heavy chainsequence of SEQ ID NO: 81 and the polynucleotide SEQ ID NO: 111 encodingthe light chain sequence of SEQ ID NO: 101.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab17 following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab17 or Fab fragmentsthereof may be produced via expression of Ab17 polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab1.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 121:

(SEQ ID NO: 131) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtaactatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatttatgatgatggtgacacatactacgctagttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatcactggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 122:

(SEQ ID NO: 132) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtaactatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatttatgatgatggtgacacatactacgctagttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatcactggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 130:

(SEQ ID NO: 140) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:141:

(SEQ ID NO: 151) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtagttacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtggtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 142:

(SEQ ID NO: 152) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtagttacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtggtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 150:

(SEQ ID NO: 160) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 134; SEQ ID NO: 136; and SEQ ID NO: 138, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 121or the variable heavy chain sequence of SEQ ID NO: 122, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 154; SEQ ID NO: 156and SEQ ID NO: 158, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 141 or the variable light chain sequence of SEQ ID NO: 142,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 133; SEQ ID NO: 135; SEQ ID NO: 137; and SEQ ID NO: 139,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 121 orthe variable heavy chain sequence of SEQ ID NO: 122, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 153; SEQ ID NO: 155; SEQID NO: 157; and SEQ ID NO: 159, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 141 or the variable light chain sequence of SEQ ID NO: 142, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 131 encoding the heavy chain sequence of SEQID NO: 121; the polynucleotide SEQ ID NO: 132 encoding the variableheavy chain sequence of SEQ ID NO: 122; the polynucleotide SEQ ID NO:151 encoding the light chain sequence of SEQ ID NO: 141; thepolynucleotide SEQ ID NO: 152 encoding the variable light chain sequenceof SEQ ID NO: 142; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 134; SEQ ID NO: 136; andSEQ ID NO: 138) of the heavy chain sequence of SEQ ID NO: 121 or thevariable heavy chain sequence of SEQ ID NO: 122; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 154; SEQ IDNO: 156; and SEQ ID NO: 158) of the light chain sequence of SEQ ID NO:141 or the variable light chain sequence of SEQ ID NO: 142;polynucleotides encoding the framework regions (SEQ ID NO: 133; SEQ IDNO: 135; SEQ ID NO: 137; and SEQ ID NO: 139) of the heavy chain sequenceof SEQ ID NO: 121 or the variable heavy chain sequence of SEQ ID NO:122; and polynucleotides encoding the framework regions (SEQ ID NO: 153;SEQ ID NO: 155; SEQ ID NO: 157; and SEQ ID NO: 159) of the light chainsequence of SEQ ID NO: 141 or the variable light chain sequence of SEQID NO: 142.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab1.H, thepolynucleotides encoding the full length Ab1.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 131 encoding theheavy chain sequence of SEQ ID NO: 121 and the polynucleotide SEQ ID NO:151 encoding the light chain sequence of SEQ ID NO: 141.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab1.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab1.H or Fab fragmentsthereof may be produced via expression of Ab1.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab2.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 161:

(SEQ ID NO: 171) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtaagtatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgatgatggcgacacatattacgctagttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 162:

(SEQ ID NO: 172) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtaagtatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgatgatggcgacacatattacgctagttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 170:

(SEQ ID NO: 180) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:181:

(SEQ ID NO: 191) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtaactacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgagggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 182:

(SEQ ID NO: 192) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtaactacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgagggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain

(SEQ ID NO: 200) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 174; SEQ ID NO: 176; and SEQ ID NO: 178, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 161or the variable heavy chain sequence of SEQ ID NO: 162, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 194; SEQ ID NO: 196and SEQ ID NO: 198, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 181 or the variable light chain sequence of SEQ ID NO: 182,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 173; SEQ ID NO: 175; SEQ ID NO: 177; and SEQ ID NO: 179,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 161 orthe variable heavy chain sequence of SEQ ID NO: 162, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 193; SEQ ID NO: 195; SEQID NO: 197; and SEQ ID NO: 199, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 181 or the variable light chain sequence of SEQ ID NO: 182, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 171 encoding the heavy chain sequence of SEQID NO: 161; the polynucleotide SEQ ID NO: 172 encoding the variableheavy chain sequence of SEQ ID NO: 162; the polynucleotide SEQ ID NO:191 encoding the light chain sequence of SEQ ID NO: 181; thepolynucleotide SEQ ID NO: 192 encoding the variable light chain sequenceof SEQ ID NO: 182; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 174; SEQ ID NO: 176; andSEQ ID NO: 178) of the heavy chain sequence of SEQ ID NO: 161 or thevariable heavy chain sequence of SEQ ID NO: 162; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 194; SEQ IDNO: 196; and SEQ ID NO: 198) of the light chain sequence of SEQ ID NO:181 or the variable light chain sequence of SEQ ID NO: 182;polynucleotides encoding the framework regions (SEQ ID NO: 173; SEQ IDNO: 175; SEQ ID NO: 177; and SEQ ID NO: 179) of the heavy chain sequenceof SEQ ID NO: 161 or the variable heavy chain sequence of SEQ ID NO:162; and polynucleotides encoding the framework regions (SEQ ID NO: 193;SEQ ID NO: 195; SEQ ID NO: 197; and SEQ ID NO: 199) of the light chainsequence of SEQ ID NO: 181 or the variable light chain sequence of SEQID NO: 182.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab2.H, thepolynucleotides encoding the full length Ab2.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 171 encoding theheavy chain sequence of SEQ ID NO: 161 and the polynucleotide SEQ ID NO:191 encoding the light chain sequence of SEQ ID NO: 181.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab2.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab2.H or Fab fragmentsthereof may be produced via expression of Ab2.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab3.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 201:

(SEQ ID NO: 211) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggttcctccctcagtaactttgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgattttggtagcacatactacgccagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding tire variable heavy chain polypeptidesequence of SEQ ID NO: 202:

(SEQ ID NO: 212) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggttcctccctcagtaactttgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgattttggtagcacatactacgccagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 210:

(SEQ ID NO: 220) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:221:

(SEQ ID NO: 231) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtgaggatattagtagtaacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaatttactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtattggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 222:

(SEQ ID NO: 232) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtgaggatattagtagtaacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaatttactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtattggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 230:

(SEQ ID NO: 240) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 214; SEQ ID NO: 216; and SEQ ID NO: 218, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 201or the variable heavy chain sequence of SEQ ID NO: 202, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 234; SEQ ID NO: 236and SEQ ID NO: 238, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 221 or the variable light chain sequence of SEQ ID NO: 222,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 213; SEQ ID NO: 215; SEQ ID NO: 217; and SEQ ID NO: 219,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 201 orthe variable heavy chain sequence of SEQ ID NO: 202, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 233; SEQ ID NO: 235; SEQID NO: 237; and SEQ ID NO: 239, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 221 or the variable light chain sequence of SEQ ID NO: 222, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 211 encoding the heavy chain sequence of SEQID NO: 201; the polynucleotide SEQ ID NO: 212 encoding the variableheavy chain sequence of SEQ ID NO: 202; the polynucleotide SEQ ID NO:231 encoding the light chain sequence of SEQ ID NO: 221; thepolynucleotide SEQ ID NO: 232 encoding the variable light chain sequenceof SEQ ID NO: 222; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 214; SEQ ID NO: 216; andSEQ ID NO: 218) of the heavy chain sequence of SEQ ID NO: 201 or thevariable heavy chain sequence of SEQ ID NO: 202; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 234; SEQ IDNO: 236; and SEQ ID NO: 238) of the light chain sequence of SEQ ID NO:221 or the variable light chain sequence of SEQ ID NO: 222;polynucleotides encoding the framework regions (SEQ ID NO: 213; SEQ IDNO: 215; SEQ ID NO: 217; and SEQ ID NO: 219) of the heavy chain sequenceof SEQ ID NO: 201 or the variable heavy chain sequence of SEQ ID NO:202; and polynucleotides encoding the framework regions (SEQ ID NO: 233;SEQ ID NO: 235; SEQ ID NO: 237; and SEQ ID NO: 239) of the light chainsequence of SEQ ID NO: 221 or the variable light chain sequence of SEQID NO: 222.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab3.H, thepolynucleotides encoding the full length Ab3.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 211 encoding theheavy chain sequence of SEQ ID NO: 201 and the polynucleotide SEQ ID NO:231 encoding the light chain sequence of SEQ ID NO: 221.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab3.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab3.H or Fab fragmentsthereof may be produced via expression of Ab3.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab4.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 241:

(SEQ ID NO: 251) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtaagcatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgatgatggtgatacatactacgctaattctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 242:

(SEQ ID NO: 252) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtaagcatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgatgatggtgatacatactacgctaattctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 250:

(SEQ ID NO: 260) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:261:

(SEQ ID NO: 271) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtagagccagtcagagcattagtgtctacctcgcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatcaggcatccaaactggcctctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggg agagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 262:

(SEQ ID NO: 272) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtagagccagtcagagcattagtgtctacctcgcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatcaggcatccaaactggcctctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 270:

(SEQ ID NO: 280) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 254; SEQ ID NO: 256; and SEQ ID NO: 258, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 241or the variable heavy chain sequence of SEQ ID NO: 242, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 274; SEQ ID NO: 276and SEQ ID NO: 278, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 261 or the variable light chain sequence of SEQ ID NO: 262,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 253; SEQ ID NO: 255; SEQ ID NO: 257; and SEQ ID NO: 259,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 241 orthe variable heavy chain sequence of SEQ ID NO: 242, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 273; SEQ ID NO: 275; SEQID NO: 277; and SEQ ID NO: 279, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 261 or the variable light chain sequence of SEQ ID NO: 262, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 251 encoding the heavy chain sequence of SEQID NO: 241; the polynucleotide SEQ ID NO: 252 encoding the variableheavy chain sequence of SEQ ID NO: 242; the polynucleotide SEQ ID NO:271 encoding the light chain sequence of SEQ ID NO: 261; thepolynucleotide SEQ ID NO: 272 encoding the variable light chain sequenceof SEQ ID NO: 262; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 254; SEQ ID NO: 256; andSEQ ID NO: 258) of the heavy chain sequence of SEQ ID NO: 241 or thevariable heavy chain sequence of SEQ ID NO: 242; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 274; SEQ IDNO: 276; and SEQ ID NO: 278) of the light chain sequence of SEQ ID NO:261 or the variable light chain sequence of SEQ ID NO: 262;polynucleotides encoding the framework regions (SEQ ID NO: 253; SEQ IDNO: 255; SEQ ID NO: 257; and SEQ ID NO: 259) of the heavy chain sequenceof SEQ ID NO: 241 or the variable heavy chain sequence of SEQ ID NO:242; and polynucleotides encoding the framework regions (SEQ ID NO: 273;SEQ ID NO: 275; SEQ ID NO: 277; and SEQ ID NO: 279) of the light chainsequence of SEQ ID NO: 261 or the variable light chain sequence of SEQID NO: 262.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab4.H, thepolynucleotides encoding the full length Ab4.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 251 encoding theheavy chain sequence of SEQ ID NO: 241 and the polynucleotide SEQ ID NO:271 encoding the light chain sequence of SEQ ID NO: 261.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab4.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab4.H or Fab fragmentsthereof may be produced via expression of Ab4.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab6.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 281:

(SEQ ID NO: 291) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattctccctcactgactatgcaatgagctgggtccgtcaggctccagggaaggggctggagtggatcggaatcattagtgatagtggtagcacatactacgctagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagagcccgagtacggctacgatgagtatggtgattgggtttctgacttatggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 282:

(SEQ ID NO: 292) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattctccctcactgactatgcaatgagctgggtccgtcaggctccagggaaggggctggagtggatcggaatcattagtgatagtggtagcacatactacgctagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagagcccgagtacggctacgatgagtatggtgattgggtttctgacttatggggccaagggaccctc gtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 290:

(SEQ ID NO: 300) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:301:

(SEQ ID NO: 311) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccactcagagcattggtaataacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatagggcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgatitigcaacttactactgtcaaagctattactatagtagtagtattacttatcataatgctttcggcggaggaaccaaggtggaaatcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 302:

(SEQ ID NO: 312) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccactcagagcattggtaataacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatagggcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgatitigcaacttactactgtcaaagctattactatagtagtagtattacttatcataatgctttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 310:

(SEQ ID NO: 320) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 294; SEQ ID NO: 296; and SEQ ID NO: 298, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 281or the variable heavy chain sequence of SEQ ID NO: 282, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 314; SEQ ID NO: 316and SEQ ID NO: 318, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 301 or the variable light chain sequence of SEQ ID NO: 302,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 293; SEQ ID NO: 295; SEQ ID NO: 297; and SEQ ID NO: 299,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 281 orthe variable heavy chain sequence of SEQ ID NO: 282, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 313; SEQ ID NO: 315; SEQID NO: 317; and SEQ ID NO: 319, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 301 or the variable light chain sequence of SEQ ID NO: 302, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 291 encoding the heavy chain sequence of SEQID NO: 281; the polynucleotide SEQ ID NO: 292 encoding the variableheavy chain sequence of SEQ ID NO: 282; the polynucleotide SEQ ID NO:311 encoding the light chain sequence of SEQ ID NO: 301; thepolynucleotide SEQ ID NO: 312 encoding the variable light chain sequenceof SEQ ID NO: 302; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 294; SEQ ID NO: 296; andSEQ ID NO: 298) of the heavy chain sequence of SEQ ID NO: 281 or thevariable heavy chain sequence of SEQ ID NO: 282; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 314; SEQ IDNO: 316; and SEQ ID NO: 318) of the light chain sequence of SEQ ID NO:301 or the variable light chain sequence of SEQ ID NO: 302;polynucleotides encoding the framework regions (SEQ ID NO: 293; SEQ IDNO: 295; SEQ ID NO: 297; and SEQ ID NO: 299) of the heavy chain sequenceof SEQ ID NO: 281 or the variable heavy chain sequence of SEQ ID NO:282; and polynucleotides encoding the framework regions (SEQ ID NO: 313;SEQ ID NO: 315; SEQ ID NO: 317; and SEQ ID NO: 319) of the light chainsequence of SEQ ID NO: 301 or the variable light chain sequence of SEQID NO: 302.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab6.H, thepolynucleotides encoding the full length Ab6.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 291 encoding theheavy chain sequence of SEQ ID NO: 281 and the polynucleotide SEQ ID NO:311 encoding the light chain sequence of SEQ ID NO: 301.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab6.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab6.H or Fab fragmentsthereof may be produced via expression of Ab6.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab7.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 321:

(SEQ ID NO: 331) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattctccctcagtagctatgcaatgagctgggtccgtcaggctccagggaaggggctggagtggatcggaatcattagtgatagtggtagcacatactacgcgagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagagcccgagtacggctacgatgactatggtgattgggtttctgacttatggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 322:

(SEQ ID NO: 332) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattctccctcagtagctatgcaatgagctgggtccgtcaggctccagggaaggggctggagtggatcggaatcattagtgatagtggtagcacatactacgcgagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagagcccgagtacggctacgatgactatggtgattgggtttctgacttatggggccaagggaccctc gtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 330:

(SEQ ID NO: 340) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:341:

(SEQ ID NO: 351) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtgattacttatcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatagggcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgatitigcaacttactactgtcaaagctattactatagtagtagtattacttatcgtaatgctttcggcggaggaaccaaggtggaaatcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 342:

(SEQ ID NO: 352) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtgattacttatcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatagggcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctattactatagtagtagtattacttatcgtaatgctttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 350:

(SEQ ID NO: 360) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 334; SEQ ID NO: 336; and SEQ ID NO: 338, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 321or the variable heavy chain sequence of SEQ ID NO: 322, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 354; SEQ ID NO: 356and SEQ ID NO: 358, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 341 or the variable light chain sequence of SEQ ID NO: 342,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 333; SEQ ID NO: 335; SEQ ID NO: 337; and SEQ ID NO: 339,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 321 orthe variable heavy chain sequence of SEQ ID NO: 322, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 353; SEQ ID NO: 355; SEQID NO: 357; and SEQ ID NO: 359, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 341 or the variable light chain sequence of SEQ ID NO: 342, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 331 encoding the heavy chain sequence of SEQID NO: 321; the polynucleotide SEQ ID NO: 332 encoding the variableheavy chain sequence of SEQ ID NO: 322; the polynucleotide SEQ ID NO:351 encoding the light chain sequence of SEQ ID NO: 341; thepolynucleotide SEQ ID NO: 352 encoding the variable light chain sequenceof SEQ ID NO: 342; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 334; SEQ ID NO: 336; andSEQ ID NO: 338) of the heavy chain sequence of SEQ ID NO: 321 or thevariable heavy chain sequence of SEQ ID NO: 322; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 354; SEQ IDNO: 356; and SEQ ID NO: 358) of the light chain sequence of SEQ ID NO:341 or the variable light chain sequence of SEQ ID NO: 342;polynucleotides encoding the framework regions (SEQ ID NO: 333; SEQ IDNO: 335; SEQ ID NO: 337; and SEQ ID NO: 339) of the heavy chain sequenceof SEQ ID NO: 321 or the variable heavy chain sequence of SEQ ID NO:322; and polynucleotides encoding the framework regions (SEQ ID NO: 353;SEQ ID NO: 355; SEQ ID NO: 357; and SEQ ID NO: 359) of the light chainsequence of SEQ ID NO: 341 or the variable light chain sequence of SEQID NO: 342.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab7.H, thepolynucleotides encoding the full length Ab7.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 331 encoding theheavy chain sequence of SEQ ID NO: 321 and the polynucleotide SEQ ID NO:351 encoding the light chain sequence of SEQ ID NO: 341.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab7.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab7.H or Fab fragmentsthereof may be produced via expression of Ab7.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab7A.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 361:

(SEQ ID NO: 371) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattctccctcagtagctatgcaatgagctgggtccgtcaggctccagggaaggggctggagtggatcggaatcattagtgatagtggtagcacatactacgcgagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagagcccgagtacggctacgatgactatggtgattgggtttctgacttatggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 362:

(SEQ ID NO: 372) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattctccctcagtagctatgcaatgagctgggtccgtcaggctccagggaaggggctggagtggatcggaatcattagtgatagtggtagcacatactacgcgagctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctagagagcccgagtacggctacgatgactatggtgattgggtttctgacttatggggccaagggaccctc gtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 370:

(SEQ ID NO: 380) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:381:

(SEQ ID NO: 391) gctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtgattacttatcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatagggcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctattactatagtagtagtattacttatcgtaatgctttcggcggaggaaccaaggtggaaatcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 382:

(SEQ ID NO: 392) gctgacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattagtgattacttatcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatagggcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctattactatagtagtagtattacttatcgtaatgctttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 390:

(SEQ ID NO: 400) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 374; SEQ ID NO: 376; and SEQ ID NO: 378, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 361or the variable heavy chain sequence of SEQ ID NO: 362, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 394; SEQ ID NO: 396and SEQ ID NO: 398, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 381 or the variable light chain sequence of SEQ ID NO: 382,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 373; SEQ ID NO: 375; SEQ ID NO: 377; and SEQ ID NO: 379,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 361 orthe variable heavy chain sequence of SEQ ID NO: 362, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 393; SEQ ID NO: 395; SEQID NO: 397; and SEQ ID NO: 399, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 381 or the variable light chain sequence of SEQ ID NO: 382, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 371 encoding the heavy chain sequence of SEQID NO: 361; the polynucleotide SEQ ID NO: 372 encoding the variableheavy chain sequence of SEQ ID NO: 362; the polynucleotide SEQ ID NO:391 encoding the light chain sequence of SEQ ID NO: 381; thepolynucleotide SEQ ID NO: 392 encoding the variable light chain sequenceof SEQ ID NO: 382; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 374; SEQ ID NO: 376; andSEQ ID NO: 378) of the heavy chain sequence of SEQ ID NO: 361 or thevariable heavy chain sequence of SEQ ID NO: 362; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 394; SEQ IDNO: 396; and SEQ ID NO: 398) of the light chain sequence of SEQ ID NO:381 or the variable light chain sequence of SEQ ID NO: 382;polynucleotides encoding the framework regions (SEQ ID NO: 373; SEQ IDNO: 375; SEQ ID NO: 377; and SEQ ID NO: 379) of the heavy chain sequenceof SEQ ID NO: 361 or the variable heavy chain sequence of SEQ ID NO:362; and polynucleotides encoding the framework regions (SEQ ID NO: 393;SEQ ID NO: 395; SEQ ID NO: 397; and SEQ ID NO: 399) of the light chainsequence of SEQ ID NO: 381 or the variable light chain sequence of SEQID NO: 382.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab7A.H, thepolynucleotides encoding the full length Ab7A.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 371 encoding theheavy chain sequence of SEQ ID NO: 361 and the polynucleotide SEQ ID NO:391 encoding the light chain sequence of SEQ ID NO: 381.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab7A.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab7A.H or Fab fragmentsthereof may be produced via expression of Ab7A.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab10.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 401:

(SEQ ID NO: 411) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtagcgctgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatttatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtagtgtctggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 402:

(SEQ ID NO: 412) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtagcgctgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatttatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtagtgtctggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 410:

(SEQ ID NO: 420) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:421:

(SEQ ID NO: 431) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtgagaacatttacaggtctttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 422:

(SEQ ID NO: 432) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtgagaacatttacaggtctttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 430:

(SEQ ID NO: 440) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 414; SEQ ID NO: 416; and SEQ ID NO: 418, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 401or the variable heavy chain sequence of SEQ ID NO: 402, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 434; SEQ ID NO: 436and SEQ ID NO: 438, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 421 or the variable light chain sequence of SEQ ID NO: 422,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 413; SEQ ID NO: 415; SEQ ID NO: 417; and SEQ ID NO: 419,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 401 orthe variable heavy chain sequence of SEQ ID NO: 402, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 433; SEQ ID NO: 435; SEQID NO: 437; and SEQ ID NO: 439, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 421 or the variable light chain sequence of SEQ ID NO: 422, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 411 encoding the heavy chain sequence of SEQID NO: 401; the polynucleotide SEQ ID NO: 412 encoding the variableheavy chain sequence of SEQ ID NO: 402; the polynucleotide SEQ ID NO:431 encoding the light chain sequence of SEQ ID NO: 421; thepolynucleotide SEQ ID NO: 432 encoding the variable light chain sequenceof SEQ ID NO: 422; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 414; SEQ ID NO: 416; andSEQ ID NO: 418) of the heavy chain sequence of SEQ ID NO: 401 or thevariable heavy chain sequence of SEQ ID NO: 402; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 434; SEQ IDNO: 436; and SEQ ID NO: 438) of the light chain sequence of SEQ ID NO:421 or the variable light chain sequence of SEQ ID NO: 422;polynucleotides encoding the framework regions (SEQ ID NO: 413; SEQ IDNO: 415; SEQ ID NO: 417; and SEQ ID NO: 419) of the heavy chain sequenceof SEQ ID NO: 401 or the variable heavy chain sequence of SEQ ID NO:402; and polynucleotides encoding the framework regions (SEQ ID NO: 433;SEQ ID NO: 435; SEQ ID NO: 437; and SEQ ID NO: 439) of the light chainsequence of SEQ ID NO: 421 or the variable light chain sequence of SEQID NO: 422.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab10.H, thepolynucleotides encoding the full length Ab10.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 411 encoding theheavy chain sequence of SEQ ID NO: 401 and the polynucleotide SEQ ID NO:431 encoding the light chain sequence of SEQ ID NO: 421.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab10.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab10.H or Fab fragmentsthereof may be produced via expression of Ab10.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab11.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 441:

(SEQ ID NO: 451) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtgcctatgacatcctctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatgtatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 442:

(SEQ ID NO: 452) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtgcctatgacatcctctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatgtatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 450:

(SEQ ID NO: 460) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:461:

(SEQ ID NO: 471) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattgatagtagcttggcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagttactatggtattggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 462:

(SEQ ID NO: 472) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattgatagtagcttggcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagttactatggtattggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain

(SEQ ID NO: 480) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 454; SEQ ID NO: 456; and SEQ ID NO: 458, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 441or the variable heavy chain sequence of SEQ ID NO: 442, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 474; SEQ ID NO: 476and SEQ ID NO: 478, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 461 or the variable light chain sequence of SEQ ID NO: 462,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 453; SEQ ID NO: 455; SEQ ID NO: 457; and SEQ ID NO: 459,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 441 orthe variable heavy chain sequence of SEQ ID NO: 442, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 473; SEQ ID NO: 475; SEQID NO: 477; and SEQ ID NO: 479, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 461 or the variable light chain sequence of SEQ ID NO: 462, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 451 encoding the heavy chain sequence of SEQID NO: 441; the polynucleotide SEQ ID NO: 452 encoding the variableheavy chain sequence of SEQ ID NO: 442; the polynucleotide SEQ ID NO:471 encoding the light chain sequence of SEQ ID NO: 461; thepolynucleotide SEQ ID NO: 472 encoding the variable light chain sequenceof SEQ ID NO: 462; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 454; SEQ ID NO: 456; andSEQ ID NO: 458) of the heavy chain sequence of SEQ ID NO: 441 or thevariable heavy chain sequence of SEQ ID NO: 442; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 474; SEQ IDNO: 476; and SEQ ID NO: 478) of the light chain sequence of SEQ ID NO:461 or the variable light chain sequence of SEQ ID NO: 462;polynucleotides encoding the framework regions (SEQ ID NO: 453; SEQ IDNO: 455; SEQ ID NO: 457; and SEQ ID NO: 459) of the heavy chain sequenceof SEQ ID NO: 441 or the variable heavy chain sequence of SEQ ID NO:442; and polynucleotides encoding the framework regions (SEQ ID NO: 473;SEQ ID NO: 475; SEQ ID NO: 477; and SEQ ID NO: 479) of the light chainsequence of SEQ ID NO: 461 or the variable light chain sequence of SEQID NO: 462.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab11.H, thepolynucleotides encoding the full length Ab11.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 451 encoding theheavy chain sequence of SEQ ID NO: 441 and the polynucleotide SEQ ID NO:471 encoding the light chain sequence of SEQ ID NO: 461.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab11.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab11.H or Fab fragmentsthereof may be produced via expression of Ab11.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab11A.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 481:

(SEQ ID NO: 491) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtgcctatgacatcctctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatgtatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 482:

(SEQ ID NO: 492) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtgcctatgacatcctctgggtccgtcaggctccagggaaggggctggagtccatcggaatgatgtatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatctggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 490:

(SEQ ID NO: 500) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:501:

(SEQ ID NO: 511) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattggtagtagcttggcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgatitigcaacttactactgtcaaagctatgaaggtagtagtagtagttactatggtattggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 502:

(SEQ ID NO: 512) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattggtagtagcttggcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctattctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgatitigcaacttactactgtcaaagctatgaaggtagtagtagtagttactatggtattggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 510:

(SEQ ID NO: 520) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 494; SEQ ID NO: 496; and SEQ ID NO: 498, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 481or the variable heavy chain sequence of SEQ ID NO: 482, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 514; SEQ ID NO: 516and SEQ ID NO: 518, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 501 or the variable light chain sequence of SEQ ID NO: 502,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 493; SEQ ID NO: 495; SEQ ID NO: 497; and SEQ ID NO: 499,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 481 orthe variable heavy chain sequence of SEQ ID NO: 482, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 513; SEQ ID NO: 515; SEQID NO: 517; and SEQ ID NO: 519, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 501 or the variable light chain sequence of SEQ ID NO: 502, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 491 encoding the heavy chain sequence of SEQID NO: 481; the polynucleotide SEQ ID NO: 492 encoding the variableheavy chain sequence of SEQ ID NO: 482; the polynucleotide SEQ ID NO:511 encoding the light chain sequence of SEQ ID NO: 501; thepolynucleotide SEQ ID NO: 512 encoding the variable light chain sequenceof SEQ ID NO: 502; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 494; SEQ ID NO: 496; andSEQ ID NO: 498) of the heavy chain sequence of SEQ ID NO: 481 or thevariable heavy chain sequence of SEQ ID NO: 482; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 514; SEQ IDNO: 516; and SEQ ID NO: 518) of the light chain sequence of SEQ ID NO:501 or the variable light chain sequence of SEQ ID NO: 502;polynucleotides encoding the framework regions (SEQ ID NO: 493; SEQ IDNO: 495; SEQ ID NO: 497; and SEQ ID NO: 499) of the heavy chain sequenceof SEQ ID NO: 481 or the variable heavy chain sequence of SEQ ID NO:482; and polynucleotides encoding the framework regions (SEQ ID NO: 513;SEQ ID NO: 515; SEQ ID NO: 517; and SEQ ID NO: 519) of the light chainsequence of SEQ ID NO: 501 or the variable light chain sequence of SEQID NO: 502.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab11A.H, thepolynucleotides encoding the full length Ab11A.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 491 encoding theheavy chain sequence of SEQ ID NO: 481 and the polynucleotide SEQ ID NO:511 encoding the light chain sequence of SEQ ID NO: 501.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab11A.H following expression ofthe full-length polynucleotides in a suitable host. In anotherembodiment of the invention, anti-ACTH antibodies such as Ab11A.H or Fabfragments thereof may be produced via expression of Ab11A.Hpolynucleotides in mammalian cells such as CHO, NSO or HEK 293 cells,fungal, insect, or microbial systems such as yeast cells (for examplediploid yeast such as diploid Pichia) and other yeast strains. SuitablePichia species include, but are not limited to, Pichia pastoris.

Antibody Ab12.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 521:

(SEQ ID NO: 531) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggatcctccctcagtgattatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatgtggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggt aaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 522:

(SEQ ID NO: 532) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggatcctccctcagtgattatgacatgatctgggtccgtcaggctccagggaaggggctggagtccatcggaatcatttatgatgatggtgacacatactacgctacttctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgtcaaaggtgtgagtaatatgtggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 530:

(SEQ ID NO: 540) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:541:

(SEQ ID NO: 551) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattggtagtagcttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatgctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 542:

(SEQ ID NO: 552) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagagcattggtagtagcttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatgctgcatccactctggcatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaagctatgatggtagtagtagtagtagttatggtgttggtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 550:

(SEQ ID NO: 560) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 534; SEQ ID NO: 536; and SEQ ID NO: 538, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 521or the variable heavy chain sequence of SEQ ID NO: 522, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 554; SEQ ID NO: 556and SEQ ID NO: 558, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 541 or the variable light chain sequence of SEQ ID NO: 542,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 533; SEQ ID NO: 535; SEQ ID NO: 537; and SEQ ID NO: 539,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 521 orthe variable heavy chain sequence of SEQ ID NO: 522, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 553; SEQ ID NO: 555; SEQID NO: 557; and SEQ ID NO: 559, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 541 or the variable light chain sequence of SEQ ID NO: 542, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 531 encoding the heavy chain sequence of SEQID NO: 521; the polynucleotide SEQ ID NO: 532 encoding the variableheavy chain sequence of SEQ ID NO: 522; the polynucleotide SEQ ID NO:551 encoding the light chain sequence of SEQ ID NO: 541; thepolynucleotide SEQ ID NO: 552 encoding the variable light chain sequenceof SEQ ID NO: 542; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 534; SEQ ID NO: 536; andSEQ ID NO: 538) of the heavy chain sequence of SEQ ID NO: 521 or thevariable heavy chain sequence of SEQ ID NO: 522; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 554; SEQ IDNO: 556; and SEQ ID NO: 558) of the light chain sequence of SEQ ID NO:541 or the variable light chain sequence of SEQ ID NO: 542;polynucleotides encoding the framework regions (SEQ ID NO: 533; SEQ IDNO: 535; SEQ ID NO: 537; and SEQ ID NO: 539) of the heavy chain sequenceof SEQ ID NO: 521 or the variable heavy chain sequence of SEQ ID NO:522; and polynucleotides encoding the framework regions (SEQ ID NO: 553;SEQ ID NO: 555; SEQ ID NO: 557; and SEQ ID NO: 559) of the light chainsequence of SEQ ID NO: 541 or the variable light chain sequence of SEQID NO: 542.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab12.H, thepolynucleotides encoding the full length Ab12.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 531 encoding theheavy chain sequence of SEQ ID NO: 521 and the polynucleotide SEQ ID NO:551 encoding the light chain sequence of SEQ ID NO: 541.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab12.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab12.H or Fab fragmentsthereof may be produced via expression of Ab12.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab13.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 561:

(SEQ ID NO: 571) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtagcggctacgacatctgctgggtccgtcaggctccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgctagctctgctaaaggccgattcaccatgtccagagacaattccaagaacaccgtgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctaagggtatttctagtatatggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtct ccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 562:

(SEQ ID NO: 572) gaggtgcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtagcggctacgacatctgctgggtccgtcaggctccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgctagctctgctaaaggccgattcaccatgtccagagacaattccaagaacaccgtgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctaagggtatttctagtatatggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 570:

(SEQ ID NO: 580) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:581:

(SEQ ID NO: 591) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatgctgcatccaagctgacatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaacatactatgatatcattgatgatggtgctactttcggcggaggaaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca acaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 582:

(SEQ ID NO: 592) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatgctgcatccaagctgacatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaacatactatgatatcattgatgatggtgctactttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 590:

(SEQ ID NO: 600) acggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 574; SEQ ID NO: 576; and SEQ ID NO: 578, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 561or the variable heavy chain sequence of SEQ ID NO: 562, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 594; SEQ ID NO: 596and SEQ ID NO: 598, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 581 or the variable light chain sequence of SEQ ID NO: 582,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 573; SEQ ID NO: 575; SEQ ID NO: 577; and SEQ ID NO: 579,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 561 orthe variable heavy chain sequence of SEQ ID NO: 562, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 593; SEQ ID NO: 595; SEQID NO: 597; and SEQ ID NO: 599, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 581 or the variable light chain sequence of SEQ ID NO: 582, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 571 encoding the heavy chain sequence of SEQID NO: 561; the polynucleotide SEQ ID NO: 572 encoding the variableheavy chain sequence of SEQ ID NO: 562; the polynucleotide SEQ ID NO:591 encoding the light chain sequence of SEQ ID NO: 581; thepolynucleotide SEQ ID NO: 592 encoding the variable light chain sequenceof SEQ ID NO: 582; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 574; SEQ ID NO: 576; andSEQ ID NO: 578) of the heavy chain sequence of SEQ ID NO: 561 or thevariable heavy chain sequence of SEQ ID NO: 562; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 594; SEQ IDNO: 596; and SEQ ID NO: 598) of the light chain sequence of SEQ ID NO:581 or the variable light chain sequence of SEQ ID NO: 582;polynucleotides encoding the framework regions (SEQ ID NO: 573; SEQ IDNO: 575; SEQ ID NO: 577; and SEQ ID NO: 579) of the heavy chain sequenceof SEQ ID NO: 561 or the variable heavy chain sequence of SEQ ID NO:562; and polynucleotides encoding the framework regions (SEQ ID NO: 593;SEQ ID NO: 595; SEQ ID NO: 597; and SEQ ID NO: 599) of the light chainsequence of SEQ ID NO: 581 or the variable light chain sequence of SEQID NO: 582.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab13.H, thepolynucleotides encoding the full length Ab13.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 571 encoding theheavy chain sequence of SEQ ID NO: 561 and the polynucleotide SEQ ID NO:591 encoding the light chain sequence of SEQ ID NO: 581.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab13.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab13.H or Fab fragmentsthereof may be produced via expression of Ab13.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab15.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 601:

(SEQ ID NO: 611) cagcagcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtgacacctacgacatgtgctgggtccgtcaggctccagggaaggggctggagtggatcggatgcatcgatactggtagtggtgacacttactacccaacctctgctaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctaagggtgtttccagtttatggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 602:

(SEQ ID NO: 620) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 610:

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:621:

(SEQ ID NO: 631) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtgaggacattgaaagcgatttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatggtgcatccactctgaagtctggagtcccatcaaggttcagcggcagtggatctggaacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaacctattatgatatggctgatgatggtgctagtttcggcggaggaaccaaggtggaaatcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca acaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 622:

(SEQ ID NO: 632) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtgaggacattgaaagcgatttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatggtgcatccactctgaagtctggagtcccatcaaggttcagcggcagtggatctggaacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaacctattatgatatggctgatgatggtgctagtttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 630:

(SEQ ID NO: 640) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 614; SEQ ID NO: 616; and SEQ ID NO: 618, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 601or the variable heavy chain sequence of SEQ ID NO: 602, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 634; SEQ ID NO: 636and SEQ ID NO: 638, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 621 or the variable light chain sequence of SEQ ID NO: 622,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 613; SEQ ID NO: 615; SEQ ID NO: 617; and SEQ ID NO: 619,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 601 orthe variable heavy chain sequence of SEQ ID NO: 602, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 633; SEQ ID NO: 635; SEQID NO: 637; and SEQ ID NO: 639, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 621 or the variable light chain sequence of SEQ ID NO: 622, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 611 encoding the heavy chain sequence of SEQID NO: 601; the polynucleotide SEQ ID NO: 612 encoding the variableheavy chain sequence of SEQ ID NO: 602; the polynucleotide SEQ ID NO:631 encoding the light chain sequence of SEQ ID NO: 621; thepolynucleotide SEQ ID NO: 632 encoding the variable light chain sequenceof SEQ ID NO: 622; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 614; SEQ ID NO: 616; andSEQ ID NO: 618) of the heavy chain sequence of SEQ ID NO: 601 or thevariable heavy chain sequence of SEQ ID NO: 602; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 634; SEQ IDNO: 636; and SEQ ID NO: 638) of the light chain sequence of SEQ ID NO:621 or the variable light chain sequence of SEQ ID NO: 622;polynucleotides encoding the framework regions (SEQ ID NO: 613; SEQ IDNO: 615; SEQ ID NO: 617; and SEQ ID NO: 619) of the heavy chain sequenceof SEQ ID NO: 601 or the variable heavy chain sequence of SEQ ID NO:602; and polynucleotides encoding the framework regions (SEQ ID NO: 633;SEQ ID NO: 635; SEQ ID NO: 637; and SEQ ID NO: 639) of the light chainsequence of SEQ ID NO: 621 or the variable light chain sequence of SEQID NO: 622.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab15.H, thepolynucleotides encoding the full length Ab15.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 611 encoding theheavy chain sequence of SEQ ID NO: 601 and the polynucleotide SEQ ID NO:631 encoding the light chain sequence of SEQ ID NO: 621.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab15.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab15.H or Fab fragmentsthereof may be produced via expression of Ab15.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Antibody Ab17.H

In one embodiment, the invention is further directed to polynucleotidesencoding antibody polypeptides having binding specificity to ACTH. Inone embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the heavy chain sequence of SEQ ID NO: 641:

(SEQ ID NO: 651) cagcagcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtagcggctacgacatctgctgggtccgtcaggctccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgccagctctgcaaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctaagggtatttctagtttatggggccaagggaccctcgtcaccgtctcgagcgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, the polynucleotides of theinvention comprise, or alternatively consist of, the followingpolynucleotide sequence encoding the variable heavy chain polypeptidesequence of SEQ ID NO: 642:

(SEQ ID NO: 652) cagcagcagcttgtggagtctgggggaggcttggtccagcctggggggtccctgagactctcctgtgcagcctctggattcaccgtcagtagcggctacgacatctgctgggtccgtcaggctccagggaaggggctggagtggatcggatgcattgatactggtagtggtaacacttactacgccagctctgcaaaaggccgattcaccatctccagagacaattccaagaacaccctgtatcttcaaatgaacagcctgagagctgaggacactgctgtgtattactgtgctaagggtatttctagtttatggggccaagggaccctcgtcaccgtctcgagc.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant heavy chain polypeptide sequence of SEQID NO: 650:

(SEQ ID NO: 660) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacgcgagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacgccagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the light chain polypeptide sequence of SEQ ID NO:661:

(SEQ ID NO: 671) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatgctgcatccaaactgacatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaacctattatgatattagtgatgatggtgctactttcggcggaggaaccaaggtggaaatcaaacgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttca acaggggagagtgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the variable light chain polypeptide sequence of SEQID NO: 662:

(SEQ ID NO: 672) gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggccagtcagaccattagtagcgacttagcctggtatcagcagaaaccaggaaaagcccctaagctcctgatctatgctgcatccaaactgacatctggagtcccatcaaggttcagcggcagtggatctggaacagaattcactctcaccatcagcagcctgcagcctgatgattttgcaacttactactgtcaaacctattatgatattagtgatgatggtgctactttcggcggaggaaccaaggtggaaatcaaacgt.

In another embodiment of the invention, polynucleotides of the inventioncomprise, or alternatively consist of, the following polynucleotidesequence encoding the constant light chain polypeptide sequence of SEQID NO: 670:

(SEQ ID NO: 680) acggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagc ttcaacaggggagagtgt.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 654; SEQ ID NO: 656; and SEQ ID NO: 658, which correspond topolynucleotides encoding the complementarity-determining regions (CDRsor hypervariable regions) of the heavy chain sequence of SEQ ID NO: 641or the variable heavy chain sequence of SEQ ID NO: 642, and/or one ormore of the polynucleotide sequences of SEQ ID NO: 674; SEQ ID NO: 676and SEQ ID NO: 678, which correspond to the complementarity-determiningregions (CDRs or hypervariable regions) of the light chain sequence ofSEQ ID NO: 661 or the variable light chain sequence of SEQ ID NO: 662,or combinations of these polynucleotide sequences. In another embodimentof the invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of polynucleotides encoding one or more of the CDRs, thevariable heavy chain and variable light chain sequences, and the heavychain and light chain sequences set forth above, including all of them.

In a further embodiment of the invention, polynucleotides encodingantibody fragments having binding specificity to ACTH comprise, oralternatively consist of, one or more of the polynucleotide sequences ofSEQ ID NO: 653; SEQ ID NO: 655; SEQ ID NO: 657; and SEQ ID NO: 659,which correspond to polynucleotides encoding the framework regions (FRsor constant regions) of the heavy chain sequence of SEQ ID NO: 641 orthe variable heavy chain sequence of SEQ ID NO: 642, and/or one or moreof the polynucleotide sequences of SEQ ID NO: 673; SEQ ID NO: 675; SEQID NO: 677; and SEQ ID NO: 679, which correspond to the frameworkregions (FRs or constant regions) of the light chain sequence of SEQ IDNO: 661 or the variable light chain sequence of SEQ ID NO: 662, orcombinations of these polynucleotide sequences. In another embodiment ofthe invention, the polynucleotides encoding the antibodies of theinvention or fragments thereof comprise, or alternatively consist of,combinations of one or more of the FRs, the variable heavy chain andvariable light chain sequences, and the heavy chain and light chainsequences set forth above, including all of them.

The invention also contemplates polynucleotide sequences including oneor more of the polynucleotide sequences encoding antibody fragmentsdescribed herein. In one embodiment of the invention, polynucleotidesencoding antibody fragments having binding specificity to ACTH comprise,or alternatively consist of, one, two, three or more, including all ofthe following polynucleotides encoding antibody fragments: thepolynucleotide SEQ ID NO: 651 encoding the heavy chain sequence of SEQID NO: 641; the polynucleotide SEQ ID NO: 652 encoding the variableheavy chain sequence of SEQ ID NO: 642; the polynucleotide SEQ ID NO:671 encoding the light chain sequence of SEQ ID NO: 661; thepolynucleotide SEQ ID NO: 672 encoding the variable light chain sequenceof SEQ ID NO: 662; polynucleotides encoding thecomplementarity-determining regions (SEQ ID NO: 654; SEQ ID NO: 656; andSEQ ID NO: 658) of the heavy chain sequence of SEQ ID NO: 641 or thevariable heavy chain sequence of SEQ ID NO: 642; polynucleotidesencoding the complementarity-determining regions (SEQ ID NO: 674; SEQ IDNO: 676; and SEQ ID NO: 678) of the light chain sequence of SEQ ID NO:661 or the variable light chain sequence of SEQ ID NO: 662;polynucleotides encoding the framework regions (SEQ ID NO: 653; SEQ IDNO: 655; SEQ ID NO: 657; and SEQ ID NO: 659) of the heavy chain sequenceof SEQ ID NO: 641 or the variable heavy chain sequence of SEQ ID NO:642; and polynucleotides encoding the framework regions (SEQ ID NO: 673;SEQ ID NO: 675; SEQ ID NO: 677; and SEQ ID NO: 679) of the light chainsequence of SEQ ID NO: 661 or the variable light chain sequence of SEQID NO: 662.

In a preferred embodiment of the invention, polynucleotides of theinvention comprise, or alternatively consist of, polynucleotidesencoding Fab (fragment antigen binding) fragments having bindingspecificity for ACTH. With respect to antibody Ab17.H, thepolynucleotides encoding the full length Ab17.H antibody comprise, oralternatively consist of, the polynucleotide SEQ ID NO: 651 encoding theheavy chain sequence of SEQ ID NO: 641 and the polynucleotide SEQ ID NO:671 encoding the light chain sequence of SEQ ID NO: 661.

Another embodiment of the invention contemplates these polynucleotidesincorporated into an expression vector for expression in mammalian cellssuch as CHO, NSO, HEK-293, or in fungal, insect, or microbial systemssuch as yeast cells such as the yeast Pichia. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris. In one embodiment ofthe invention described herein (infra), Fab fragments may be produced byenzymatic digestion (e.g., papain) of Ab17.H following expression of thefull-length polynucleotides in a suitable host. In another embodiment ofthe invention, anti-ACTH antibodies such as Ab17.H or Fab fragmentsthereof may be produced via expression of Ab17.H polynucleotides inmammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, ormicrobial systems such as yeast cells (for example diploid yeast such asdiploid Pichia) and other yeast strains. Suitable Pichia speciesinclude, but are not limited to, Pichia pastoris.

Host cells and vectors comprising said polynucleotides are alsocontemplated.

The invention further contemplates vectors comprising the polynucleotidesequences encoding the variable heavy and light chain polypeptidesequences, as well as the individual complementarity-determining regions(CDRs, or hypervariable regions), as set forth herein, as well as hostcells comprising said vector sequences. In one embodiment of theinvention, the host cell is a yeast cell. In another embodiment of theinvention, the yeast host cell belongs to the genus Pichia.

Exemplary Embodiments of the Subject Disclosure

B-Cell Screening and Isolation

The subject anti-ACTH antibodies and variants thereof, especiallychimerized variants were obtained from clonal populations of B cellsderived from rabbits which had been immunized with human ACTH. Such Bcell screening and isolation methods have been previously described andare disclosed in U.S. Provisional Application No. 61/791,755 filed Mar.15, 2013, and U.S. Ser. No. 14/217,594 filed Mar. 18, 2014, which eachof which is expressly incorporated by reference herein.

Methods of Humanizing Antibodies

In another embodiment, the present invention contemplates methods forhumanizing antibody heavy and light chains. Methods for humanizingantibody heavy and light chains which may be applied to anti-ACTHantibodies are taught, for example, in U.S. patent applicationpublication no. US 2009/0022659 to Olson et al., and in U.S. Pat. No.7,935,340 to Garcia-Martinez et al., the disclosures of each of whichare herein incorporated by reference in their entireties.

Methods of Producing Antibodies and Fragments Thereof

In another embodiment, the present invention contemplates methods forproducing anti-ACTH antibodies and fragments thereof. Methods forproducing anti-ACTH antibodies and fragments thereof secreted frompolyploid, preferably diploid or tetraploid strains of mating competentyeast are taught, for example, in U.S. patent application publicationno. US 2009/0022659 to Olson et al., and in U.S. Pat. No. 7,935,340 toGarcia-Martinez et al., the disclosures of each of which are hereinincorporated by reference in their entireties. A preferred yeast formanufacture of antibodies is of the genus Pichia, and more preferablyPichia pastoris. However, antibodies according to the inventionpotentially may be made in other yeast such as other mating competentyeast of the Saccharomycetaceae family, which includes the generaArxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera;Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea;Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces. Othertypes of yeast potentially useful for making antibody proteins accordingto the invention include Yarrowia; Rhodosporidium; Candida; Hansenula;Filobasium; Sporidiobolus; Bullera; Leucosporidium and Filobasidella.

Other methods of producing antibodies are well known to those ofordinary skill in the art. For example, methods of producing chimericantibodies are now well known in the art (See, for example, U.S. Pat.No. 4,816,567 to Cabilly et al.; Morrison et al., PNAS. USA, 81:8651-55(1984); Neuberger, M. S. et al., Nature, 314:268-270 (1985); Boulianne,G. L. et al., Nature, 312:643-46 (1984), the disclosures of each ofwhich are herein incorporated by reference in their entireties).

Likewise, other methods of producing humanized antibodies are now wellknown in the art (See, for example, U.S. Pat. Nos. 5,530,101, 5,585,089,5,693,762, and 6,180,370 to Queen et al; U.S. Pat. Nos. 5,225,539 and6,548,640 to Winter; U.S. Pat. Nos. 6,054,297, 6,407,213 and 6,639,055to Carter et al; U.S. Pat. No. 6,632,927 to Adair; Jones, P. T. et al,Nature, 321:522-525 (1986); Reichmann, L., et al, Nature, 332:323-327(1988); Verhoeyen, M, et al, Science, 239:1534-36 (1988), thedisclosures of each of which are herein incorporated by reference intheir entireties).

Antibody polypeptides of the invention having ACTH binding specificitymay also be produced by constructing, using conventional techniques wellknown to those of ordinary skill in the art, an expression vectorcontaining an operon and a DNA sequence encoding an antibody heavy chainin which the DNA sequence encoding the CDRs required for antibodyspecificity is derived from a non-human cell source, preferably a rabbitB-cell source, while the DNA sequence encoding the remaining parts ofthe antibody chain is derived from a human cell source.

A second expression vector is produced using the same conventional meanswell known to those of ordinary skill in the art, said expression vectorcontaining an operon and a DNA sequence encoding an antibody light chainin which the DNA sequence encoding the CDRs required for antibodyspecificity is derived from a non-human cell source, preferably a rabbitB-cell source, while the DNA sequence encoding the remaining parts ofthe antibody chain is derived from a human cell source.

The expression vectors are transfected into a host cell by conventiontechniques well known to those of ordinary skill in the art to produce atransfected host cell, said transfected host cell cultured byconventional techniques well known to those of ordinary skill in the artto produce said antibody polypeptides.

The host cell may be co-transfected with the two expression vectorsdescribed above, the first expression vector containing DNA encoding anoperon and a light chain-derived polypeptide and the second vectorcontaining DNA encoding an operon and a heavy chain-derived polypeptide.The two vectors contain different selectable markers, but preferablyachieve substantially equal expression of the heavy and light chainpolypeptides. Alternatively, a single vector may be used, the vectorincluding DNA encoding both the heavy and light chain polypeptides. Thecoding sequences for the heavy and light chains may comprise cDNA,genomic DNA, or both.

Host cells which potentially may be used to express the subject antibodypolypeptides may include bacterial cells such as E. coli, or eukaryoticcells such as P. pastoris, other yeast cells, fungi, insect cells,mammalian cells, and plant cells. In one embodiment of the invention, amammalian cell of a well-defined type may be for this purpose, such as amyeloma cell, a Chinese hamster ovary (CHO) cell line, a NSO cell line,or a HEK293 cell line.

The general methods by which the vectors may be constructed,transfection methods required to produce the host cell and culturingmethods required to produce the antibody polypeptides from said hostcells all include conventional techniques. Although preferably the cellline used to produce the antibody is a mammalian cell line, any othersuitable cell line, such as a bacterial cell line such as an E.coli-derived bacterial strain, or a yeast cell line, may alternativelybe used.

Similarly, once produced the antibody polypeptides may be purifiedaccording to standard procedures in the art, such as for examplecross-flow filtration, ammonium sulphate precipitation, affinity columnchromatography and the like.

The antibody polypeptides described herein may also be used for thedesign and synthesis of either peptide or non-peptide mimetics thatwould be useful for the same therapeutic applications as the antibodypolypeptides of the invention. See, for example, Saragobi et al,Science, 253:792-795 (1991), the contents of which are hereinincorporated by reference in its entirety.

Screening Assays

The invention also includes screening assays designed to assist in theidentification of diseases and disorders associated with ACTH insubjects exhibiting symptoms of an ACTH associated disease or disorder.

In some embodiments, the antibody is used as a diagnostic tool. Theantibody can be used to assay the amount of ACTH present in a sampleand/or subject. As will be appreciated by one of skill in the art, suchantibodies need not be neutralizing antibodies. In some embodiments, thediagnostic antibody is not a neutralizing antibody. In some embodiments,the diagnostic antibody binds to a different epitope than theneutralizing antibody binds to. In some embodiments, the two antibodiesdo not compete with one another.

In some embodiments, the antibodies disclosed herein are used orprovided in an assay kit and/or method for the detection of ACTH inmammalian tissues or cells in order to screen/diagnose for a disease ordisorder associated with changes in levels of ACTH. The kit comprises anantibody that binds ACTH and means for indicating the binding of theantibody with ACTH, if present, and optionally ACTH protein levels.Various means for indicating the presence of an antibody can be used.For example, fluorophores, other molecular probes, or enzymes can belinked to the antibody and the presence of the antibody can be observedin a variety of ways. The method for screening for such disorders caninvolve the use of the kit, or simply the use of one of the disclosedantibodies and the determination of whether the antibody binds to ACTHin a sample. As will be appreciated by one of skill in the art, high orelevated levels of ACTH will result in larger amounts of the antibodybinding to ACTH in the sample. Thus, degree of antibody binding can beused to determine how much ACTH is in a sample. Subjects or samples withan amount of ACTH that is greater than a predetermined amount (e.g., anamount or range that a person without an ACTH-related disorder wouldhave) can be characterized as having an ACTH-mediated disorder. In someembodiments, the antibody is administered to a subject taking a statin,in order to determine if the statin has affected the amount of ACTH inthe subject.

The invention is also directed to a method of in vivo imaging whichdetects the presence of cells which express ACTH comprisingadministering a diagnostically effective amount of a diagnosticcomposition. Said in vivo imaging is useful for the detection or imagingof ACTH expressing cells or organs, for example, and can be useful aspart of a planning regimen for the design of an effective treatmentprotocol.

The present invention further provides for a kit for detecting bindingof an anti-ACTH antibody of the invention to ACTH. In particular, thekit may be used to detect the presence of an ACTH specifically reactivewith an anti-ACTH antibody of the invention or an immunoreactivefragment thereof. The kit may also include an antibody bound to asubstrate, a secondary antibody reactive with the antigen and a reagentfor detecting a reaction of the secondary antibody with the antigen.Such a kit may be an ELISA kit and can comprise the substrate, primaryand secondary antibodies when appropriate, and any other necessaryreagents such as detectable moieties, enzyme substrates, and colorreagents, for example as described herein. The diagnostic kit may alsobe in the form of an immunoblot kit. The diagnostic kit may also be inthe form of a chemiluminescent kit (Meso Scale Discovery, Gaithersburg,Md.). The diagnostic kit may also be a lanthanide-based detection kit(PerkinElmer, San Jose, Calif.).

A skilled clinician would understand that a biological sample includes,but is not limited to, sera, plasma, urine, saliva, mucous, pleuralfluid, synovial fluid and spinal fluid.

Methods of Ameliorating or Reducing Symptoms of, or Treating, orPreventing, Diseases and Disorders Associated with, ACTH

In another embodiment of the invention, anti-ACTH antibodies describedherein, or fragments thereof, are useful for ameliorating or reducingthe symptoms of, or treating, or preventing, diseases and disordersassociated with ACTH. As mentioned, these conditions include, by way ofexample, ACTH-driven hypercortisolism, acute coronary syndrome, acuteheart failure, anxiety disorders, atherosclerosis, atrial fibrillation,cachexia, cancer (such as Cushing's Syndrome resulting from ectopic ACTHexpression, e.g., in small cell lung cancer, non-small cell lung cancer(NSCLC), pancreatic carcinoma, neural tumors, or thymoma), cardiacconditions, cardiac fibrosis, cardiovascular disorders, chronic renalfailure, chronic stress syndrome, cognitive dysfunction, Alzheimer'sdisease, congenital adrenal hyperplasia (CAH), Classical CAH,Nonclassical CAH, familial glucocorticoid deficiency (FGD), congestiveheart failure, Conn's syndrome, coronary heart diseases, Cushing'sDisease, Cushing's Syndrome, depression, diabetes, endothelialdysfunction, exercise intolerance, familial hyperaldosteronism,fibrosis, galactorrhea, heart failure, hyperaldosteronism,hypercortisolemia, hypertension, hyperinsulinemia, hypokalemia, impairedcardiac function, increased formation of collagen, inflammation,metabolic syndrome, muscle atrophy, conditions associated with muscleatrophy, myocardiac fibrosis, nephropathy, obesity, post-myocardialinfarction, primary hyperaldosteronism, remodeling followinghypertension, renal failure, restenosis, secondary hyperaldosteronism,sleep apnea, stress related conditions, or syndrome X.

Anti-ACTH antibodies described herein, or fragments thereof, as well ascombinations, can also be administered in a therapeutically orprophylactically effective amount to subjects in need of treatment orprevention of diseases and disorders associated with ACTH in the form ofa pharmaceutical or diagnostic composition as described in greaterdetail below.

In another embodiment of the invention, anti-ACTH antibodies describedherein, or fragments thereof, with or without a second agent, are usefulfor ameliorating or reducing the symptoms of, or treating, orpreventing, disorders that relate to, involve, or can be influenced byvaried ACTH, corticosterone, cortisol, and/or aldosterone levels. Insome embodiments, the antibody or antibody fragment according to theinvention is useful in reducing the risk of, symptoms of, treating, orpreventing congenital adrenal hyperplasia (CAH), Classical CAH,Nonclassical CAH, familial glucocorticoid deficiency (FGD), ACTH-drivenhypercortisolism (Cushing's Disease and/or Cushing's Syndrome), obesity,diabetes, sleep disorders such as, e.g., sleep apnea, narcolepsy andinsomnia, depression, anxiety disorders, cancer (such as Cushing'sSyndrome resulting from ectopic ACTH expression, e.g., in small celllung cancer, non-small cell lung cancer (NSCLC), pancreatic carcinoma,neural tumors, or thymoma), muscle atrophy, hypertension,hyperinsulinemia, cognitive dysfunction, Alzheimer's disease,galactorrhea, stress related conditions, impaired cardiac function,exercise intolerance, heart failure and other cardiac conditions,metabolic syndrome, hyperaldosteronism including primaryhyperaldosteronism (such as Conn's syndrome) secondaryhyperaldosteronism, and familial hyperaldosteronism; or Allgrovesyndrome, bilateral adrenalectomy, or Nelson's syndrome.

In further exemplary embodiments the present invention provides formonitoring and/or additional treatment of a subject (including a humanor non-human animal subject) to which an anti-ACTH antibody of thepresent disclosure has been administered. Said monitoring may includemonitoring of cortisol levels, and monitoring of other symptoms ofadrenal insufficiency, such as primary and/or secondary adrenalinsufficiency. Said additional treatment may include glucocorticoidand/or mineralocorticoid replacement, and/or treatment of other symptomsof adrenal insufficiency. Said glucocorticoid and/or mineralocorticoidreplacement may be in an amount effective to treat adrenalinsufficiency, or a symptom thereof. Without intent to be limited bytheory, it is believed that in vivo antagonism of ACTH, such as thatresulting from administration of an anti-ACTH antibody, may result inthe development of one or more symptoms associated with adrenalinsufficiency. Monitoring and treatment of adrenal insufficiency isreviewed in Arit and Allolio, “Adrenal Insufficiency,” Lancet361:1881-1893 (2003) and also described in The Merck Manual of Diagnosisand Therapy, 19th edition, Merck & Company, 19201192, each of which ishereby incorporated by reference in its entirety.

After administration of an anti-ACTH antibody, a subject may bemonitored for symptoms associated with adrenal insufficiency, which mayinclude symptoms associated with primary and/or secondary adrenalinsufficiency. Such symptoms may include decreased cortisol levelsand/or hypoglycemia after fasting (thought to result from decreasedgluconeogenesis). Decreased cortisol levels may be detected as morningcortisol levels decreased to lower than a reference range (such as165-680 nmol/L), e.g., to less than 165 nmol/L or less than 100 nmol/L.Said symptoms associated with primary and/or secondary adrenalinsufficiency may include low serum sodium (e.g., <135 mEq/L), highserum potassium (e.g., >5 mEq/L), low carbonate (e.g., 15 to 20 mEq/L),and/or high BUN. Said symptoms may be compared to baseline measurementsand observations prior to administration of an anti-ACTH antibody.

Glucocorticoid replacement therapy may include administration ofhydrocortisone (such as at a dosage of between 15-25 mg daily, between3-125 mg daily, or higher or lower dosages), cortisone acetate (such asat a dosage of 25-37.5 mg daily, between 5-200 mg daily, or higher orlower dosages), dexamethasone (such as at a dosage of 0.03 mg/kg/day to0.15 mg/kg/day, between 0.005 mg/kg/day to 0.75 mg/kg/day, or higher orlower dosages), or other glucocorticoid administration. Theglucocorticoid may be a glucocorticoid that does not interfere withmonitoring of cortisol levels (such as dexamethasone). Preferably saidglycocorticoid is administered in an amount effective to treat adrenalinsufficiency, or a symptom thereof. Glucocorticoid replacement therapymay optionally be divided among multiple equal or unequal daily doses,e.g., half to two-third dose in the morning (such as immediately afterrising) and the remainder in one or more subsequent doses.Glucocorticoid replacement therapy may be administered in atime-released form including but not limited to a single dailytime-released administration. Glucocorticoid replacement therapy mayalso be delivered using an implantable medical device which may beadapted to release the drug in a controlled manner over an extendedperiod of time, which may mimic the normal diurnal pattern of cortisollevels. Glucocorticoid replacement therapy may be adjusted as indicatedby any signs or symptoms suggestive of over- or under-replacement. Onesuch sign would be based on the ability of the subject to cope withdaily stress (e.g., determined based on a fasting glucose test).Glucocorticoid dosage may be determined adjusted based on individualsubject attributes, symptoms, and responses. Dosage may be increased inresponse to or anticipation of stress. For example, in advance ofstrenuous physical activity, a subject's dosage of hydrocortisone may beincreased by approximately 5-10 mg per day. Further, for subjects undersevere physical stress, such as fever, the hydrocortisone dosage may beincreased, e.g., doubled. For major surgery, trauma, and diseases thatrequire monitoring and intensive care, the hydrocortisone dosage may beincreased to 100-150 mg per 24 hours. For more minor or moderatesurgical stress lower dosages such as up to 25-75 mg hydrocortisone per24 hours may be administered. Parenteral administration ofglucocorticoids and/or mineralocorticoids may be performed in instancesof vomiting or diarrhea.

Treatment of a subject may further include mineralocorticoidreplacement, e.g., 0.05-0.2 mg fludrocortisone administered per day,such as in a single daily dose or other dosage form. Alternative oradditional mineralocorticoids may be administered. Dosage may bedetermined adjusted based on individual subject attributes, symptoms,and responses. Further, effects of glucocorticoids and other treatmentsmay be taken into consideration in selecting a dosage. For example, forhydrocortisone dosages of 50 mg per day (or higher), mineralocorticoidreplacement may be reduced or omitted as such dosages of hydrocortisonecan have similar effects as 0.1 mg fludrocortisone. Mineralocorticoidreplacement may be initiated or altered based upon signs or symptomsindicative of a need therefore, or indicative of over- orunder-replacement, e.g., as indicated by monitoring of blood pressure,peripheral edema, serum sodium, serum potassium, plasma renin activity,carbonate, and/or BUN.

Subjects may further be monitored and/or treated for adrenal crisis.Signs and symptoms of adrenal crisis include one or more of thefollowing: sudden penetrating pain in the legs, lower back or abdomen;confusion; psychosis; slurred speech; severe lethargy; convulsions;fever; hyperkalemia; hypercalcemia; hypoglycemia; hyponatremia;hypotension; hypothyroid (low T4 level); severe vomiting and diarrhea,potentially resulting in dehydration; and/or syncope. In acute adrenalcrisis, subjects may be immediately administered 100 mg hydrocortisone(typically intravenously) followed by 100-200 mg hydrocortisone per 24hours. Adrenal crisis may also be treated by administration of 4-5 mgdexamethasone. A higher or lower dosage may be administered, e.g.,immediate administration of between 20 and 250 mg hydrocortisone, orbetween 1 and 25 mg of dexamethasone, or a higher or lower dosage.Continuous large infusions of physiological saline may be performed,such as at a rate of 1 liter per hour or another amount effective totreat the patient. Continuous cardiac monitoring may be performed, withtreatment optionally being modified appropriately as the subject'scondition develops.

Administration

In one embodiment of the invention, the anti-ACTH antibodies describedherein, or ACTH binding fragments thereof, as well as combinations ofsaid antibodies or antibody fragments, are administered to a subject ata concentration of between about 0.1 and 100.0 mg/kg of body weight ofrecipient subject. In a preferred embodiment of the invention, theanti-ACTH antibodies described herein, or ACTH binding fragmentsthereof, as well as combinations of said antibodies or antibodyfragments, are administered to a subject at a concentration of about 0.4mg/kg of body weight of recipient subject. In a preferred embodiment ofthe invention, the anti-ACTH antibodies described herein, or ACTHbinding fragments thereof, as well as combinations of said antibodies orantibody fragments, are administered to a recipient subject with afrequency of once every twenty-six weeks or less, such as once everysixteen weeks or less, once every eight weeks or less, once every fourweeks or less, once every two weeks or less, once every week or less, oronce daily or less.

Fab fragments may be administered every two weeks or less, every week orless, once daily or less, multiple times per day, and/or every fewhours. In one embodiment of the invention, a subject receives Fabfragments of 0.1 mg/kg to 40 mg/kg per day given in divided doses of 1to 6 times a day, or in a sustained release form, effective to obtaindesired results.

It is to be understood that the concentration of the antibody or Fabadministered to a given subject may be greater or lower than theexemplary administration concentrations set forth above.

A person of skill in the art would be able to determine an effectivedosage and frequency of administration through routine experimentation,for example guided by the disclosure herein and the teachings inGoodman, L. S., Gilman, A., Brunton, L. L., Lazo, J. S., & Parker, K. L.(2006). Goodman & Gilman's the pharmacological basis of therapeutics.New York: McGraw-Hill; Howland, R. D., Mycek, M. J., Harvey, R. A.,Champe, P. C., & Mycek, M. J. (2006). Pharmacology. Lippincott'sillustrated reviews. Philadelphia: Lippincott Williams & Wilkins; andGolan, D. E. (2008). Principles of pharmacology: the pathophysiologicbasis of drug therapy. Philadelphia, Pa., [etc.]: Lippincott Williams &Wilkins.

In another embodiment of the invention, the anti-ACTH antibodiesdescribed herein, or ACTH binding fragments thereof, as well ascombinations of said antibodies or antibody fragments, are administeredto a subject in a pharmaceutical formulation.

A “pharmaceutical composition” refers to a chemical or biologicalcomposition suitable for administration to a mammal. Such compositionsmay be specifically formulated for administration via one or more of anumber of routes, including but not limited to buccal, epicutaneous,epidural, inhalation, intraarterial, intracardial,intracerebroventricular, intradermal, intramuscular, intranasal,intraocular, intraperitoneal, intraspinal, intrathecal, intravenous,oral, parenteral, rectally via an enema or suppository, subcutaneous,subdermal, transdermal, and transmucosal. In addition, administrationcan occur by means of injection, powder, liquid, gel, drops, or othermeans of administration.

In one embodiment of the invention, the anti-ACTH antibodies describedherein, or ACTH binding fragments thereof, as well as combinations ofsaid antibodies or antibody fragments, may be optionally administered incombination with one or more active agents. Such active agents includeketoconazole (Nizoral®), aminoglutethimide (Cytadren®), metyrapone(Metopirone®), mitotane (Lysodren®) etomidate (Amidate®), cyproheptadine(Periactin® or Peritol®), valproic acid (Depakote®), cabergoline(Dostinex®), pasireotide (Signifor®), rosiglitazone (Avandia®),conivaptan (Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985),satavaptan (SR121463, planned trade name Aquilda®), mifepristone(Korlym®), armodafinil (Nuvigil®) and modafinil (Provigil®). Additionalexemplary active agents that may be administered in combination with thesubject antibodies or fragments include without limitation thereto oneor more of: Accupril (quinapril), Aceon (perindopril), Adalat, AdalatCC, Aldactone (spironolactone), aldosterone receptor blockers,alpha-adrenergic receptor blockers, alpha-glucosidase inhibitors, Altace(ramipril), Alteplase, aminoglutethimide (Cytadren®), amiodarone,angiotensin converting enzyme (ACE) Inhibitors, angiotensin II receptorantagonists, Angiotensin II receptor blockers (ARBs), antiarrhythmics,anti-cholesterol drugs, anti-clotting agents, antidiabetogenic drugs,anti-hypertensive agents, antiplatelet drugs, ApoA-1 mimics, aspirin,Atacand (candesartan), Avapro (irbesartan), beta blockers,beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR, CETPinhibitors, conivaptan (Vaprisol®), Cordarone (amiodarone), Coreg(carvedilol), Covera-HS, Cozaar (losartan), cyproheptadine (Periactin®or Peritol®), Demadex (torsemide), digoxin, Dilacor XR, Dilatrate-SR,Diltia XT, Diovan (valsartan), dipeptidyl peptidase-4 inhibitors,diuretics, Dobutrex (dobutamine), drugs that suppress ACTH secretion,drugs that suppress cortisol secretion, dual angiotensin convertingenzyme/neutral endopeptidase (ACE/NEP) inhibitors, endothelinantagonists, endothelin receptor blockers, Esidrix(hydrochlorothiazide), etomidate (Amidate®), Fragmin, gemfibrozil(Lopid, Gemcor), glucocorticoid receptor antagonists, heart failuredrugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine (Questran),IMDUR (isosorbide mononitrate), Inderal (propranolol), inhibitors of aNa—K-ATPase membrane pump, inhibitors of steroidogenesis, insulintherapies, Iso-Bid, Isonate, Isoptin, Isoptin SR, Isorbid (isosorbidedinitrate), Isordil, Isotrate, ketoconazole (Nizoral®), Lasix(furosemide), lixivaptan (VPA-985), Lopressor, Lotensin (benazepril),Lovenox, Mavik (trandolapril), meglitinides, metyrapone (Metopirone®),Micardis (telmisartan), mifepristone (Korlym®), mitotane (Lysodren®),Monopril (fosinopril), neutral endopeptidase (NEP) inhibitors,Normodyne, Norvasc (amlodipine), obesity-reducing agents, Omacor,pantethine, pasireotide (Signifor®), Plendil (felodipine), PPAR-gammaagonists, Primacor (milrinone), Prinivil, Procanbid (procainamide),Procardia, Procardia XL (nifedipine), renin inhibitors, Reteplase,rosiglitazone (Avandia®), satavaptan (SR121463, planned trade nameAquilda®), Sectral (acebutolol), somatostatin analogs, Sorbitrate(isosorbide dinitrate), statins, Streptokinase, Sular (nisoldipine),sulfonylurea, Tambocor (flecainide), Tenecteplase, Tenormin (atenolol),thiazolidinediones, Tiazac (diltiazem), Tissue plasminogen activator(TPA), tolvaptan (OPC-41061), Toprol-XL (metoprolol), Trandate(labetalol), Univasc (moexipril), Urokinase, valproic acid (Depakote®),vaptans, Vascor (bepridil), vasodilators, Vasodilators, vasopressinantagonists, Vasotec (enalapril), Verelan, Verelan PM (verapamil),warfarin (Coumadin), Zaroxolyn (metolazone), Zebeta (bisoprolol), orZestril (lisinopril). Further exemplary active agents include one ormore corticosteroids, including glucocorticoids and/ormineralocorticoids (including agents having one or both ofglucocorticoid and/or mineralocorticoid activity), such as cortisol(hydrocortisone), dexamethasone, cortisone, prednisone, prednisolone,methylprednisolone, dexamethasone, betamethasone, triamcinolone,beclometasone, fludrocortisone (e.g., fludrocortisone acetate),deoxycorticosterone (e.g., deoxycorticosterone acetate (DOCA)), and/oraldosterone. Any suitable combination of these active agents is alsocontemplated.

A “pharmaceutical excipient” or a “pharmaceutically acceptableexcipient” is a carrier, usually a liquid, in which an activetherapeutic agent is formulated. In one embodiment of the invention, theactive therapeutic agent is a humanized antibody described herein, orone or more fragments thereof. The excipient generally does not provideany pharmacological activity to the formulation, though it may providechemical and/or biological stability, and release characteristics.Exemplary formulations can be found, for example, in Remington'sPharmaceutical Sciences, 19^(th) Ed., Grennaro, A., Ed., 1995 which isincorporated by reference.

As used herein “pharmaceutically acceptable carrier” or “excipient”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents that arephysiologically compatible. In one embodiment, the carrier is suitablefor parenteral administration. Alternatively, the carrier can besuitable for intravenous, intraperitoneal, or intramuscularadministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the pharmaceutical compositions of the invention iscontemplated. Supplementary active compounds can also be incorporatedinto the compositions.

Pharmaceutical compositions typically must be sterile and stable underthe conditions of manufacture and storage. The invention contemplatesthat the pharmaceutical composition is present in lyophilized form. Thecomposition can be formulated as a solution, microemulsion, liposome, orother ordered structure suitable to high drug concentration. The carriercan be a solvent or dispersion medium containing, for example, water,ethanol, polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycol), and suitable mixtures thereof. The inventionfurther contemplates the inclusion of a stabilizer in the pharmaceuticalcomposition. The proper fluidity can be maintained, for example, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants.

In many cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol or sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, monostearate salts andgelatin. Moreover, the alkaline polypeptide can be formulated in atime-release formulation, for example in a composition which includes aslow release polymer. The active compounds can be prepared with carriersthat will protect the compound against rapid release, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, polylactic acid and polylactic, polyglycoliccopolymers (PLG). Many methods for the preparation of such formulationsare known to those skilled in the art.

For each of the recited embodiments, the compounds can be administeredby a variety of dosage forms. Any biologically-acceptable dosage formknown to persons of ordinary skill in the art, and combinations thereof,are contemplated. Examples of such dosage forms include, withoutlimitation, reconstitutable powders, elixirs, liquids, solutions,suspensions, emulsions, powders, granules, particles, microparticles,dispersible granules, cachets, inhalants, aerosol inhalants, patches,particle inhalants, implants, depot implants, injectables (includingsubcutaneous, intramuscular, intravenous, and intradermal), infusions,and combinations thereof.

Certain teachings related to humanization of rabbit-derived monoclonalantibodies and preferred sequence modifications to maintain antigenbinding affinity were disclosed in International Application No.PCT/US2008/064421, corresponding to International Publication No.WO/2008/144757, entitled “Novel Rabbit Antibody Humanization Methods andHumanized Rabbit Antibodies”, filed May 21, 2008, the disclosure ofwhich is herein incorporated by reference in its entirety.

Certain teachings related to producing antibodies or fragments thereofusing mating competent yeast and corresponding methods were disclosed inU.S. patent application Ser. No. 11/429,053, filed May 8, 2006, (U.S.Patent Application Publication No. US2006/0270045), the disclosure ofwhich is herein incorporated by reference in its entirety.

Veterinary Uses of the Subject Antibodies

The present disclosure additionally provides the use of the subjectantibodies in non-human animals. The working examples herein demonstratethat the subject antibodies bind within a region of human ACTH that isconserved among animal species including dog, cat, and horse. A fragmentof ACTH containing this conserved epitope sequence (ACTH 1-24) canactivate ACTH receptors, and the subject antibodies are demonstratedherein to inhibit receptor activation by this fragment. Based on theseand other results presented herein, it is expected that the antibodiesof the invention will be therapeutically effective for antagonizing ACTHin vivo in these and other animal species. Thus, antibodies or antibodyfragments comprising one or more, or all, of the CDRs of any one of theantibodies disclosed herein (e.g., Ab13, Ab15, Ab17, Ab1.H, Ab2.H,Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H,Ab13.H, Ab15.H, and Ab17.H, preferably Ab13.H) may be effective to treata condition associated with ACTH in a non-human animal.

In exemplary embodiments, the disclosure provides a therapeutic methodcomprising administering an antibody or antibody fragment comprising oneor more, or all, of the CDRs of any one of the anti-ACTH antibodiesdisclosed herein (e.g., Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H, preferably Ab13.H) to a non-human animal in need thereof.

In exemplary embodiments, the disclosure provides a therapeuticcomposition comprising an antibody or antibody fragment comprising oneor more, or all, of the CDRs of any one of the anti-ACTH antibodiesdisclosed herein (e.g., Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H, preferably Ab13.H) which is adapted for administration to anon-human animal in need thereof.

In exemplary embodiments, the disclosure provides a comprising anantibody or antibody fragment comprising one or more, or all, of theCDRs of any one of the anti-ACTH antibodies disclosed herein (e.g.,Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H,Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H, preferablyAb13.H) for use in the treatment of a non-human animal in need thereof.

Said antibody or fragment may be modified to reduce the potential immunereaction of said animal. For example, said antibody may be a chimericantibody comprising the variable light and/or variable heavy domain ofany one of the anti-ACTH antibodies disclosed herein (e.g., Ab13, Ab15,Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H,Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H, preferably Ab13.H) incombination with a constant domain sequence of the respective animalspecies (such as dog, cat, or horse). Said antibody or fragment maycomprise an antibody fragment, such as scFvs, Fab fragments, Fab′fragments, monovalent antibody fragments, and F(ab′)₂ fragments. Saidantibody or fragment may comprise a species-ized antibody (e.g.,caninized, felinized, or equinized antibody for cats, dogs, or horses,respectively) produced by a process analogous to humanization, whereinone or more framework sequences or framework residues are replaced byframework sequences or residues contained within endogenous frameworksequences of antibodies of the respective species.

Said animal species may be a species in which endogenous ACTH isconserved, e.g., having the same sequence as human ACTH, or having up toone, two, three, four, or five sequence differences from human ACTH orfrom human ACTH 1-24. For example, the ACTH of said species may have oneor more, or all, of the epitope binding residues identified in theexamples herein that are the same as the residues in human ACTH, orhaving conservative substitutions relative to the corresponding residuesin human ACTH. Preferably the administered anti-ACTH antibody is able tobind to ACTH of said animal species and antagonize activation of an ACTHreceptor in said animal species.

Additional Exemplary Embodiments of the Invention

Additional exemplary embodiments of the invention are set forth in thefollowing clauses.

Clause 1A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment that specifically binds to a linear or conformationalepitope(s) and/or competes for binding to the same linear orconformational epitope(s) on human ACTH as an anti-human ACTH antibodyselected from the group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H,Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H,Ab13.H, Ab15.H, and Ab17.H.

Clause 2A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment of Clause 1A, which specifically binds to the samelinear or conformational epitope(s) and/or competes for binding to thesame linear or conformational epitope(s) on human ACTH as Ab13.H.

Clause 3A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment of Clause 1A, which specifically binds to the samelinear or conformational epitope(s) on human ACTH as an anti-human ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 4A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment of Clause 1A, which specifically binds to the samelinear or conformational epitope(s) on human ACTH as Ab13.H.

Clause 5A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment of Clause 1A, wherein said epitope(s) is identifiedusing a binding assay that detects the binding of said anti-human ACTHantibody or antibody fragment to one or more peptides in a library ofoverlapping linear peptide fragments that span the full length of humanACTH.

Clause 6A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment of Clause 1A, wherein said epitope is identified usingalanine scanning.

Clause 7A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment that contains at least 2 complementarity determiningregions (CDRs) of an anti-human ACTH antibody selected from the groupconsisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H.

Clause 8A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment according to Clause 7A, which contains at least 3 CDRsof an anti-ACTH antibody selected from the group consisting of Ab13,Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H,Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 9A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment according to Clause 7A, which contains at least 4 CDRsof an anti-ACTH antibody selected from the group consisting of Ab13,Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H,Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 10A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment according to Clause 7A, which contains at least 5 CDRsof an anti-ACTH antibody selected from the group consisting of Ab13,Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H,Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 11A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment according to Clause 7A, which contains all 6 CDRs ofan anti-ACTH antibody selected from the group consisting of Ab13, Ab15,Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H,Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 12A. A human, humanized or chimerized anti-human ACTH antibody orantibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:4; a CDR2 sequence consisting of SEQ ID NO:6; and a CDR3 sequenceconsisting of SEQ ID NO:8; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:24; a CDR2 sequence consisting of SEQ ID NO:26; and a CDR3sequence consisting of SEQ ID NO:28.

Clause 13A. A anti-human ACTH antibody or antibody fragment according toClause 12A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:2 and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:22.

Clause 14A. An anti-human ACTH antibody or antibody fragment accordingto Clause 12A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:2, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:22.

Clause 15A. An anti-human ACTH antibody or antibody fragment accordingto Clause 12A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO: 1, and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:21.

Clause 16A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:44; a CDR2 sequence consisting of SEQ ID NO:46; and a CDR3sequence consisting of SEQ ID NO:48; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:64; a CDR2 sequence consisting of SEQ ID NO:66; and a CDR3sequence consisting of SEQ ID NO:68.

Clause 17A. An anti-human ACTH antibody or antibody fragment accordingto Clause 16A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:42, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:62.

Clause 18A. An anti-human ACTH antibody or antibody fragment accordingto Clause 16A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:42, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:62.

Clause 19A. An anti-human ACTH antibody or antibody fragment accordingto Clause 16A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:41, and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:61.

Clause 20A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:84; a CDR2 sequence consisting of SEQ ID NO:86; and a CDR3sequence consisting of SEQ ID NO:88; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO: 104; a CDR2 sequence consisting of SEQ ID NO: 106; and a CDR3sequence consisting of SEQ ID NO: 108.

Clause 21A. An anti-human ACTH antibody or antibody fragment accordingto Clause 20A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:82 and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:102.

Clause 22A. An anti-human ACTH antibody or antibody fragment accordingto Clause 20A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:82, and/or

(b) a variable light chain having the amino acid sequence of SEQ ID NO:102.

Clause 23A. An anti-human ACTH antibody or antibody fragment accordingto Clause 20A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:81, and/or

(b) a light chain having the amino acid sequence of SEQ ID NO: 101.

Clause 24A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO: 124; a CDR2 sequence consisting of SEQ ID NO: 126; and a CDR3sequence consisting of SEQ ID NO: 128; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO: 144; a CDR2 sequence consisting of SEQ ID NO: 146; and a CDR3sequence consisting of SEQ ID NO: 148.

Clause 25A. An anti-human ACTH antibody or antibody fragment accordingto Clause 24A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:122 and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:142.

Clause 26A. An anti-human ACTH antibody or antibody fragment accordingto Clause 24A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ ID NO:122, and/or

(b) a variable light chain having the amino acid sequence of SEQ ID NO:142.

Clause 27A. An anti-human ACTH antibody or antibody fragment accordingto Clause 24A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO: 121,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO: 141.

Clause 28A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO: 164; a CDR2 sequence consisting of SEQ ID NO: 166; and a CDR3sequence consisting of SEQ ID NO: 168; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:184; a CDR2 sequence consisting of SEQ ID NO: 186; and a CDR3sequence consisting of SEQ ID NO: 188.

Clause 29A. An anti-human ACTH antibody or antibody fragment accordingto Clause 28A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:162, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:182.

Clause 30A. An anti-human ACTH antibody or antibody fragment accordingto Clause 28A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ ID NO:162, and/or

(b) a variable light chain having the amino acid sequence of SEQ ID NO:182.

Clause 31A. An anti-human ACTH antibody or antibody fragment accordingto Clause 28A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO: 161,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO: 181.

Clause 32A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:204; a CDR2 sequence consisting of SEQ ID NO:206; and a CDR3sequence consisting of SEQ ID NO:208; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:224; a CDR2 sequence consisting of SEQ ID NO:226; and a CDR3sequence consisting of SEQ ID NO:228.

Clause 33A. An anti-human ACTH antibody or antibody fragment accordingto Clause 32A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:202 and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:222.

Clause 34A. An anti-human ACTH antibody or antibody fragment accordingto Clause 32A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:202, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:223.

Clause 35A. An anti-human ACTH antibody or antibody fragment accordingto Clause 32A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:201,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:221.

Clause 36A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:244; a CDR2 sequence consisting of SEQ ID NO:246; and a CDR3sequence consisting of SEQ ID NO:248; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:264; a CDR2 sequence consisting of SEQ ID NO:266; and a CDR3sequence consisting of SEQ ID NO:268.

Clause 37A. An anti-human ACTH antibody or antibody fragment accordingto Clause 36A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:242 and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:262.

Clause 38A. An anti-human ACTH antibody or antibody fragment accordingto Clause 36A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:242, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:262.

Clause 39A. An anti-human ACTH antibody or antibody fragment accordingto Clause 36A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:241,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:261.

Clause 40A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:284; a CDR2 sequence consisting of SEQ ID NO:286; and a CDR3sequence consisting of SEQ ID NO:288; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:304; a CDR2 sequence consisting of SEQ ID NO:306; and a CDR3sequence consisting of SEQ ID NO:308.

Clause 41A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:282, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:302.

Clause 42A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:282, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:302.

Clause 43A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:281,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:301.

Clause 40.1A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:324; a CDR2 sequence consisting of SEQ ID NO:326; and a CDR3sequence consisting of SEQ ID NO:328; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:344; a CDR2 sequence consisting of SEQ ID NO:346; and a CDR3sequence consisting of SEQ ID NO:348.

Clause 41.1A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.1A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:322, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:342.

Clause 42.1A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.1A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:322, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:342.

Clause 43.1A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.1A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:321,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:341.

Clause 40.2A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:364; a CDR2 sequence consisting of SEQ ID NO:366; and a CDR3sequence consisting of SEQ ID NO:368; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:384; a CDR2 sequence consisting of SEQ ID NO:386; and a CDR3sequence consisting of SEQ ID NO:388.

Clause 41.2A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.2A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:362, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:382.

Clause 42.2A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.2A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:362, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:382.

Clause 43.2A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.2A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:361,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:381.

Clause 40.3A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:404; a CDR2 sequence consisting of SEQ ID NO:406; and a CDR3sequence consisting of SEQ ID NO:408; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:424; a CDR2 sequence consisting of SEQ ID NO:426; and a CDR3sequence consisting of SEQ ID NO:428.

Clause 41.3A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.3A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:402, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:422.

Clause 42.3A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.3A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:402, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:422.

Clause 43.3A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.3A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:401,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:421.

Clause 40.4A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:444; a CDR2 sequence consisting of SEQ ID NO:446; and a CDR3sequence consisting of SEQ ID NO:448; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:464; a CDR2 sequence consisting of SEQ ID NO:466; and a CDR3sequence consisting of SEQ ID NO:468.

Clause 41.4A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.4A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:442, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:462.

Clause 42.4A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.4A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:442, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:462.

Clause 43.4A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.4A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:441,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:461.

Clause 40.5A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:484; a CDR2 sequence consisting of SEQ ID NO:486; and a CDR3sequence consisting of SEQ ID NO:488; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:504; a CDR2 sequence consisting of SEQ ID NO:506; and a CDR3sequence consisting of SEQ ID NO:508.

Clause 41.5A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.5A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:482, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:502.

Clause 42.5A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.5A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:482, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:502.

Clause 43.5A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.5A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:481,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:501.

Clause 40.6A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:524; a CDR2 sequence consisting of SEQ ID NO:526; and a CDR3sequence consisting of SEQ ID NO:528; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:544; a CDR2 sequence consisting of SEQ ID NO:546; and a CDR3sequence consisting of SEQ ID NO:548.

Clause 41.6A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.6A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:522, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:542.

Clause 42.6A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.6A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:522, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:542.

Clause 43.6A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.6A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:521,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:541.

Clause 40.7A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:564; a CDR2 sequence consisting of SEQ ID NO:566; and a CDR3sequence consisting of SEQ ID NO:568; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:584; a CDR2 sequence consisting of SEQ ID NO:586; and a CDR3sequence consisting of SEQ ID NO:588.

Clause 41.7A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.7A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:562, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:582.

Clause 42.7A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.7A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:562, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:582.

Clause 43.7A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.7A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:561,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:581.

Clause 40.8A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:604; a CDR2 sequence consisting of SEQ ID NO:606; and a CDR3sequence consisting of SEQ ID NO:608; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:624; a CDR2 sequence consisting of SEQ ID NO:626; and a CDR3sequence consisting of SEQ ID NO:628.

Clause 41.8A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.8A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:602, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:622.

Clause 42.8A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.8A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:602, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:622.

Clause 43.8A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.8A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:601,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:621.

Clause 40.9A. An human, humanized or chimerized anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1A-11A, whichcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:644; a CDR2 sequence consisting of SEQ ID NO:646; and a CDR3sequence consisting of SEQ ID NO:648; and/or

(b) a variable light chain comprising a CDR1 sequence consisting of SEQID NO:664; a CDR2 sequence consisting of SEQ ID NO:666; and a CDR3sequence consisting of SEQ ID NO:668.

Clause 41.9A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.9A, which comprises:

(a) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:642, and/or

(b) a variable light chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:662.

Clause 42.9A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.9A, which comprises:

(a) a variable heavy chain having the amino acid sequence of SEQ IDNO:642, and/or

(b) a variable light chain having the amino acid sequence of SEQ IDNO:662.

Clause 43.9A. An anti-human ACTH antibody or antibody fragment accordingto Clause 40.9A, which comprises:

(a) a heavy chain having the amino acid sequence of SEQ ID NO:641,and/or

(b) a light chain having the amino acid sequence of SEQ ID NO:661.

Clause 44A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-43.9A, wherein the antibody or antibody fragment isselected from the group consisting of scFvs, camelbodies, nanobodies,IgNAR, Fab fragments, Fab′ fragments, MetMab like antibodies, monovalentantibody fragments, and F(ab′)₂ fragments.

Clause 45A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-44A, wherein the antibody or antibody fragmentsubstantially or entirely lacks N-glycosylation and/or O-glycosylation.

Clause 46A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-45A, wherein the antibody or antibody fragment comprises ahuman constant domain.

Clause 47A. The anti-human ACTH antibody or antibody fragment of Clause46A, wherein the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody.

Clause 48A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-47A, wherein the antibody or antibody fragment comprisesan Fc region that has been modified to alter at least one of effectorfunction, half-life, proteolysis, or glycosylation.

Clause 49A. The anti-human ACTH antibody or antibody fragment of Clause48A, wherein the Fc region contains one or more mutations that alters oreliminates N- and/or O-glycosylation.

Clause 50A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-49A, wherein the antibody or antibody fragment is ahumanized antibody or antibody fragment.

Clause 51A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-50A, wherein the antibody or antibody fragment binds toACTH with a binding affinity (K_(D)) of less than or equal to 5×10⁻⁵ M,10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M,10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, or 10⁻¹³ M, e.g., as determined by surface plasmon resonance(e.g., BIAcore®) at 250 or 37° C.

Clause 52A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-51A, wherein the antibody or antibody fragment binds toACTH with a binding affinity (K_(D)) of less than or equal to 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, or 10⁻¹² M.

Clause 53A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-52A, which binds to ACTH with an off-rate (k_(d)) of lessthan or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻⁵ s⁻¹, or 10⁻⁵ s⁻¹.

Clause 54A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-53A, wherein the antibody or antibody fragment is directlyor indirectly attached to a detectable label or therapeutic agent.

Clause 55A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-54A, which when administered to a human subject inhibitsor neutralizes at least one biological effect elicited by ACTH.

Clause 56A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-55A, which neutralizes or inhibits ACTH activation ofMC2R.

Clause 57A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-55A, which neutralizes or inhibits ACTH activation of atleast one of MC1R, MC2R, MC3R, MC4R and MC5R or any combination thereof.

Clause 58A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-55A, which neutralizes or inhibits ACTH activation of eachof MC2R, MC3R and MC4R.

Clause 59A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-55A, which inhibits ACTH-induced cortisol, corticosteroneand/or aldosterone secretion.

Clause 60A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-55A, which when administered to a human subject reducesplasma cortisol, aldosterone and/or corticosterone levels.

Clause 61A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-60A, wherein the antibody or antibody fragment is capableof inhibiting the binding of ACTH to a MCR.

Clause 62A. The anti-human ACTH antibody or antibody fragment of Clause61A, wherein the MCR is at least one of MC1R, MC2R, MC3R, MC4R and MC5R;at least one of MC2R, MC3R, and MC4R; each of MC2R, MC3R, and MC4R; oreach of MC1R, MC2R, MC3R, MC4R and MC5R.

Clause 63A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-62A, wherein the antibody or antibody fragment binds toACTH with a K_(D) that is less than about 100 nM.

Clause 64A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-62A, which binds to ACTH with a K_(D) that is less thanabout 100 pM.

Clause 65A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-62A, which binds to ACTH with a K_(D) that is less thanabout 50 pM.

Clause 66A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-62A, which binds to ACTH with a K_(D) that is less thanabout 25 pM.

Clause 67A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-62A, which binds to ACTH with a K_(D) that is betweenabout 10 pM and about 100 pM.

Clause 68A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-62A, which binds to ACTH with a K_(D) that is less thanabout 40 nM.

Clause 69A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-68A, which has stronger affinity for ACTH₁₋₃₉ as comparedto alpha-MSH or CLIP and/or does not bind to alpha-MSH.

Clause 70A. The anti-human ACTH antibody or antibody fragment of Clause69A, wherein the affinity of said antibody or antibody fragment toACTH₁₋₃₉ is at least 10-fold, 100-fold, 1000-fold or more stronger thanthe affinity of said antibody or antibody fragment to alpha-MSH or CLIP(i.e., the K_(D) for ACTH is numerically lower than the K_(D) foralpha-MSH or CLIP by at least 10-fold, 100-fold, 1000-fold or more).

Clause 71A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-70A, wherein the antibody or antibody fragment is attachedto at least one effector moiety.

Clause 72A. The anti-human ACTH antibody or antibody fragment of Clause71A, wherein effector moiety comprises a chemical linker.

Clause 73A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-72A, wherein the antibody or antibody fragment is attachedto one or more detectable moieties.

Clause 74A. The anti-human ACTH antibody or antibody fragment of Clause73A, wherein detectable moiety comprises a fluorescent dye, enzyme,substrate, bioluminescent material, radioactive material,chemiluminescent moiety, or mixtures thereof.

Clause 75A. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1A-74A, wherein the antibody or antibody fragment is attachedto one or more functional moieties.

Clause 76A. An anti-idiotypic antibody produced against an anti-humanACTH antibody or antibody fragment according to any one of Clauses1A-75A, which optionally, neutralizes one or more biological effects ofthe anti-human ACTH antibody to which it binds.

Clause 77A. A method of using the anti-idiotypic antibody of Clause 76Aor another antibody that specifically binds said anti-human ACTHantibody to monitor the in vivo levels of said anti-ACTH antibody orantibody fragment in a subject or to neutralize said anti-ACTH antibodyin a subject being administered said anti-ACTH antibody or antibodyfragment or a method of using the anti-idiotypic antibody of Clause 76Aor another antibody that specifically binds said anti-human ACTHantibody to neutralize the in vivo effects of said antibody in a subjectin need thereof.

Clause 78A. A composition suitable for therapeutic, prophylactic, or adiagnostic use comprising a therapeutically, prophylactically ordiagnostically effective amount of at least one anti-human ACTH antibodyor antibody fragment or anti-idiotypic antibody according to any one ofClauses 1A-76A.

Clause 79A. The composition of Clause 78A, which is suitable forsubcutaneous administration.

Clause 80A. The composition of Clause 78A, which is suitable forintravenous administration.

Clause 81A. The composition of Clause 78A, which is lyophilized.

Clause 82A. The composition of any one of Clauses 78A-81A, furthercomprising a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative, or mixture thereof.

Clause 83A. The composition of any one of Clauses 78A-82A, furthercomprising another active agent.

Clause 84A. The composition of Clause 83A, wherein (i) the other activeagent is selected from the group consisting of ketoconazole (Nizoral®),aminoglutethimide (Cytadren®), metyrapone (Metopirone®), mitotane(Lysodren®) etomidate (Amidate®), cyproheptadine (Periactin® orPeritol®), valproic acid (Depakote®), cabergoline (Dostinex®),pasireotide (Signifor®), rosiglitazone (Avandia®), conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), satavaptan(SR121463, planned trade name Aquilda®), mifepristone (Korlym®),armodafinil (Nuvigil®) and modafinil (Provigil®), or wherein the otheractive agent is selected from the group consisting of: Accupril(quinapril), Aceon (perindopril), Adalat, Adalat CC, Aldactone(spironolactone), aldosterone receptor blockers, alpha-adrenergicreceptor blockers, alpha-glucosidase inhibitors, Altace (ramipril),Alteplase, aminoglutethimide (Cytadren®), amiodarone, angiotensinconverting enzyme (ACE) Inhibitors, angiotensin II receptor antagonists,Angiotensin II receptor blockers (ARBs), antiarrhythmics,anti-cholesterol drugs, anti-clotting agents, antidiabetogenic drugs,anti-hypertensive agents, antiplatelet drugs, ApoA-1 mimics, aspirin,Atacand (candesartan), Avapro (irbesartan), beta blockers,beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR, CETPinhibitors, conivaptan (Vaprisol®), Cordarone (amiodarone), Coreg(carvedilol), Covera-HS, Cozaar (losartan), cyproheptadine (Periactin®or Peritol®), Demadex (torsemide), digoxin, Dilacor XR, Dilatrate-SR,Diltia XT, Diovan (valsartan), dipeptidyl peptidase-4 inhibitors,diuretics, Dobutrex (dobutamine), drugs that suppress ACTH secretion,drugs that suppress cortisol secretion, dual angiotensin convertingenzyme/neutral endopeptidase (ACE/NEP) inhibitors, endothelinantagonists, endothelin receptor blockers, Esidrix(hydrochlorothiazide), etomidate (Amidate®), Fragmin, gemfibrozil(Lopid, Gemcor), glucocorticoid receptor antagonists, heart failuredrugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine (Questran),IMDUR (isosorbide mononitrate), Inderal (propranolol), inhibitors of aNa—K-ATPase membrane pump, inhibitors of steroidogenesis, insulintherapies, Iso-Bid, Isonate, Isoptin, Isoptin SR, Isorbid (isosorbidedinitrate), Isordil, Isotrate, ketoconazole (Nizoral®), Lasix(furosemide), lixivaptan (VPA-985), Lopressor, Lotensin (benazepril),Lovenox, Mavik (trandolapril), meglitinides, metyrapone (Metopirone®),Micardis (telmisartan), mifepristone (Korlym®), mitotane (Lysodren®),Monopril (fosinopril), neutral endopeptidase (NEP) inhibitors,Normodyne, Norvasc (amlodipine), obesity-reducing agents, Omacor,pantethine, pasireotide (Signifor®), Plendil (felodipine), PPAR-gammaagonists, Primacor (milrinone), Prinivil, Procanbid (procainamide),Procardia, Procardia XL (nifedipine), renin inhibitors, Reteplase,rosiglitazone (Avandia®), satavaptan (SR121463, planned trade nameAquilda®), Sectral (acebutolol), somatostatin analogs, Sorbitrate(isosorbide dinitrate), statins, Streptokinase, Sular (nisoldipine),sulfonylurea, Tambocor (flecainide), Tenecteplase, Tenormin (atenolol),thiazolidinediones, Tiazac (diltiazem), Tissue plasminogen activator(TPA), tolvaptan (OPC-41061), Toprol-XL (metoprolol), Trandate(labetalol), Univasc (moexipril), Urokinase, valproic acid (Depakote®),vaptans, Vascor (bepridil), vasodilators, Vasodilators, vasopressinantagonists, Vasotec (enalapril), Verelan, Verelan PM (verapamil),warfarin (Coumadin), Zaroxolyn (metolazone), Zebeta (bisoprolol), andZestril (lisinopril), and/or (ii) the other active agent is selectedfrom the group consisting of: corticosteroids, glucocorticoids,mineralocorticoids, cortisol (hydrocortisone), dexamethasone, cortisone,prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone,fludrocortisone acetate, deoxycorticosterone, deoxycorticosteroneacetate (DOCA), and aldosterone.

Clause 85A. The composition of any one of Clauses 79A-84A, which islyophilized, stabilized and/or formulated for administration byinjection.

Clause 86A. An isolated nucleic acid sequence or nucleic acid sequencesencoding an anti-human ACTH antibody or antibody fragment oranti-idiotypic antibody according to any one of Clauses 1A-76A.

Clause 87A. A vector or vectors containing the isolated nucleic acidsequence or sequences of Clause 86A.

Clause 88A. A host cell comprising the isolated nucleic acid sequence orsequences of Clause 87A or the vector or vectors of Clause 87A.

Clause 89A. The host cell of Clause 88A, which is a mammalian,bacterial, fungal, yeast, avian or insect cell.

Clause 90A. The host cell of Clause 89A, which is a filamentous fungi ora yeast.

Clause 91A. The host cell of Clause 90A, wherein the yeast is selectedfrom the from the following genera: Arxiozyma; Ascobotryozyma;Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia;Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia;Saccharomyces; Saturnispora; Tetrapisispora; Torulaspora; Williopsis;and Zygosaccharomyces.

Clause 92A. The host cell of Clause 91A, which is the yeast genus isPichia.

Clause 93A. The host cell of Clause 92A, wherein the species of Pichiais selected from Pichia pastoris, Pichia methanolica and Hansenulapolymorpha (Pichia angusta).

Clause 94A. A method of expressing an anti-human ACTH antibody orantibody fragment comprising culturing the host cell of any one ofClauses 89A-93A under conditions that provide for expression of saidantibody or antibody fragment.

Clause 95A. The method of Clause 94A, wherein the host cell is apolyploid yeast culture that stably expresses and secretes into theculture medium at least 10⁻²⁵ mg/liter of said antibody or antibodyfragment.

Clause 96A. The method of Clause 95A, wherein said polyploid yeast ismade by a method that comprises:

(i) introducing at least one expression vector containing one or moreheterologous polynucleotides encoding said antibody operably linked to apromoter and a signal sequence into a haploid yeast cell;

(ii) producing by mating or spheroplast fusion a polyploid yeast fromsaid first and/or second haploid yeast cell;

(iii) selecting polyploid yeast cells that stably express said antibody;and

(iv) producing stable polyploid yeast cultures from said polyploid yeastcells that stably express said antibody into the culture medium.

Clause 97A. The method of Clause 96A, wherein said yeast is of the genusPichia.

Clause 98A. A method for blocking, inhibiting or neutralizing one ormore biological effects associated with ACTH comprising administering toa subject in need thereof an effective amount of an anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment thatspecifically binds to the same linear or conformational epitope(s)and/or competes for binding to the same linear or conformationalepitope(s) on human ACTH as an anti-human ACTH antibody selected fromthe group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H.

Clause 99A. A method for treating or preventing a condition associatedwith elevated ACTH levels in a subject, comprising administering to asubject in need thereof an effective amount of an anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment thatspecifically binds to the same linear or conformational epitope(s)and/or competes for binding to the same linear or conformationalepitope(s) on human ACTH as an anti-human ACTH antibody selected fromthe group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H.

Clause 100A. A method for treating or preventing a condition associatedwith elevated cortisol, aldosterone or corticosterone levels in asubject, comprising administering to the subject in need thereof aneffective amount of an anti-human adrenocorticotrophic hormone (“ACTH”)antibody or antibody fragment that specifically binds to the same linearor conformational epitope(s) and/or competes for binding to the samelinear or conformational epitope(s) on human ACTH as an anti-human ACTHselected from the group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H,Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H,Ab13.H, Ab15.H, and Ab17.H.

Clause 101A. The method of any one of Clauses 97A-100A, wherein (i) thecondition is selected from the group consisting of ACTH-drivenhypercortisolism (Cushing's Disease and/or Cushing's Syndrome), obesity,diabetes, Parkinson's disease, sleep disorders, e.g., insomnia, sleepapnea, and narcolepsy, depression, anxiety disorders, cancer (such asCushing's Syndrome resulting from ectopic ACTH expression, e.g., insmall cell lung cancer, non-small cell lung cancer (NSCLC), pancreaticcarcinoma, neural tumors, or thymoma), muscle atrophy, hypertension,hyperinsulinemia, cognitive dysfunction, Alzheimer's disease,galactorrhea, stress related conditions, impaired cardiac function,exercise intolerance, heart failure and other cardiac conditions,metabolic syndrome, hyperaldosteronism, Conn's syndrome and familialhyperaldosteronism, or (ii) wherein the condition comprises congenitaladrenal hyperplasia (CAH), Classical CAH, or Nonclassical CAH or (iii)wherein the condition comprises familial glucocorticoid deficiency (FGD)or (iv) wherein the condition comprises Allgrove syndrome, bilateraladrenalectomy, or Nelson's syndrome.

Clause 102A. A method for neutralizing ACTH-induced MCR signaling,comprising administering to a subject in need thereof an effectiveamount of an anti-human adrenocorticotrophic hormone (“ACTH”) antibodyor antibody fragment that specifically binds to the same linear orconformational epitope(s) and/or competes for binding to the same linearor conformational epitope(s) on human ACTH as an anti-human ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 103A. A method for inhibiting ACTH-induced cortisol, aldosteroneor corticosterone secretion, comprising administering to a subject inneed thereof an effective amount of an anti-human adrenocorticotrophichormone (“ACTH”) antibody or antibody fragment that specifically bindsto the same linear or conformational epitope(s) and/or competes forbinding to the same linear or conformational epitope(s) on human ACTH asan anti-human ACTH antibody selected from the group consisting of Ab13,Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H,Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 104A. A method for reducing ACTH-induced plasma cortisol,aldosterone or corticosterone levels in a subject in need thereof,comprising administering to a subject in need thereof an effectiveamount of a human, humanized or chimerized anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment thatspecifically binds to the same linear or conformational epitope(s)and/or competes for binding to the same linear or conformationalepitope(s) on human ACTH as an anti-human ACTH antibody selected fromthe group consisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H,Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H,and Ab17.H.

Clause 105A. The method of any one of Clauses 98A-104A, wherein theantibody is a human, humanized or chimerized anti-ACTH antibody orantibody fragment.

Clause 106A. The method of any one of Clauses 98A-105A, wherein theantibody or antibody fragment substantially does not interact with(bind) a polypeptide consisting of: (i) the 13 N-terminal amino acidresidues of ACTH (ACTH₁₋₁₃) and/or alpha-MSH, or (ii) the 22 C-terminalamino acid residues of ACTH (ACTH₁₈₋₃₉).

Clause 107A. The method of any one of Clauses 98A-106A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentinhibits the binding of ACTH to a MCR.

Clause 108A. The method of Clause 107A, wherein the MCR is selected fromthe group consisting of MC1R, MC2R, MC3R, MC4R and MC5R.

Clause 109A. The method of any one of Clauses 98A-108A, wherein saidepitope(s) is identified using a binding assay that detects the bindingof said anti-human ACTH antibody or antibody fragment to one or morepeptides in a library of overlapping linear peptide fragments that spanthe full length of human ACTH.

Clause 110A. The method of any one of Clauses 98-109A, which contains atleast 2 complementarity determining regions (CDRs) of an anti-human ACTHantibody selected from the group consisting of Ab13, Ab15, Ab17, Ab1.H,Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H,Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 111A. The method of any one of Clauses 98A-110A, which containsat least 3 CDRs of an anti-ACTH antibody selected from the groupconsisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H.

Clause 112A. The method of any one of Clauses 98A-110A, which containsat least 4 CDRs of an anti-ACTH antibody selected from the groupconsisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H.

Clause 113A. The method of any one of Clauses 98A-110A, which containsat least 5 CDRs of an anti-ACTH antibody selected from the groupconsisting of Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H,Ab7.H, Ab7A.H, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, andAb17.H.

Clause 114A. The method of any one of Clauses 98A-110A, which containsall 6 CDRs of an anti-ACTH antibody selected from the group consistingof Ab13, Ab15, Ab17, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab7A.H,Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H.

Clause 115A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:4; a CDR2 sequence consisting of SEQ ID NO:6; and a CDR3 sequenceconsisting of SEQ ID NO:8; and/or a variable light chain comprising aCDR1 sequence consisting of SEQ ID NO:24; a CDR2 sequence consisting ofSEQ ID NO:26; and a CDR3 sequence consisting of SEQ ID NO:28;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:2; and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:22;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:2; and/or a variable light chain having the amino acid sequence ofSEQ ID NO:22; or

(d) a heavy chain having the amino acid sequence of SEQ ID NO: 1, and/ora light chain having the amino acid sequence of SEQ ID NO:21.

Clause 116A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:44; a CDR2 sequence consisting of SEQ ID NO:46; and a CDR3sequence consisting of SEQ ID NO:48, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:64; a CDR2 sequenceconsisting of SEQ ID NO:66; and a CDR3 sequence consisting of SEQ IDNO:68;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:42, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:62;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:42, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:62; or

(d) a heavy chain having the amino acid sequence of SEQ ID NO:41, and/ora light chain having the amino acid sequence of SEQ ID NO:61.

Clause 117A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:84; a CDR2 sequence consisting of SEQ ID NO:86; and a CDR3sequence consisting of SEQ ID NO:88, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 104; a CDR2 sequenceconsisting of SEQ ID NO: 106; and a CDR3 sequence consisting of SEQ IDNO: 108;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:82, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 102;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:82, and/or a variable light chain having the amino acid sequence ofSEQ ID NO: 102; or

(d) a heavy chain having the amino acid sequence of SEQ ID NO:81, and/ora light chain having the amino acid sequence of SEQ ID NO:101.

Clause 118A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO: 124; a CDR2 sequence consisting of SEQ ID NO: 126 and a CDR3sequence consisting of SEQ ID NO: 128, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO: 144; a CDR2 sequenceconsisting of SEQ ID NO: 146; and a CDR3 sequence consisting of SEQ IDNO: 148;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:122 and/or a variable light chain comprising an amino acid sequence withat least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO: 142;

(c) a variable heavy chain having the amino acid sequence of SEQ ID NO:122, and/or a variable light chain having the amino acid sequence of SEQID NO: 142; or

(d) a heavy chain having the amino acid sequence of SEQ ID NO:121,and/or a light chain having the amino acid sequence of SEQ ID NO: 141.

Clause 119A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO: 164; a CDR2 sequence consisting of SEQ ID NO: 166; and a CDR3sequence consisting of SEQ ID NO: 168, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:184; a CDR2 sequenceconsisting of SEQ ID NO: 186; and a CDR3 sequence consisting of SEQ IDNO: 188;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO:162, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO: 182;

(c) a variable heavy chain having the amino acid sequence of SEQ ID NO:162, and/or a variable light chain having the amino acid sequence of SEQID NO: 182; or

(d) a heavy chain having the amino acid sequence of SEQ ID NO:161,and/or a light chain having the amino acid sequence of SEQ ID NO: 181.

Clause 120A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:204; a CDR2 sequence consisting of SEQ ID NO:206; and a CDR3sequence consisting of SEQ ID NO:208, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:224; a CDR2 sequenceconsisting of SEQ ID NO:226; and a CDR3 sequence consisting of SEQ IDNO:228;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:202 and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:222;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:202, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:222; or

(d) a heavy chain having the amino acid sequence of SEQ ID NO:201,and/or a light chain having the amino acid sequence of SEQ ID NO:221.

Clause 121A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:244; a CDR2 sequence consisting of SEQ ID NO:246; and a CDR3sequence consisting of SEQ ID NO:248, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:264; a CDR2 sequenceconsisting of SEQ ID NO:266; and a CDR3 sequence consisting of SEQ IDNO:268;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:242, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:262;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:242, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:262;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:241,and/or a light chain having the amino acid sequence of SEQ ID NO:261.

Clause 122A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:284; a CDR2 sequence consisting of SEQ ID NO:286; and a CDR3sequence consisting of SEQ ID NO:288, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:304; a CDR2 sequenceconsisting of SEQ ID NO:306; and a CDR3 sequence consisting of SEQ IDNO:308;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:282, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:302;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:282, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:302;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:281,and/or a light chain having the amino acid sequence of SEQ ID NO:301.

Clause 122.1A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:324; a CDR2 sequence consisting of SEQ ID NO:326; and a CDR3sequence consisting of SEQ ID NO:328, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:344; a CDR2 sequenceconsisting of SEQ ID NO:346; and a CDR3 sequence consisting of SEQ IDNO:348;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:322, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:342;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:322, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:342;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:321,and/or a light chain having the amino acid sequence of SEQ ID NO:341.

Clause 122.2A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:364; a CDR2 sequence consisting of SEQ ID NO:366; and a CDR3sequence consisting of SEQ ID NO:368, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:384; a CDR2 sequenceconsisting of SEQ ID NO:386; and a CDR3 sequence consisting of SEQ IDNO:388;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:362, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:382;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:362, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:382;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:361,and/or a light chain having the amino acid sequence of SEQ ID NO:381.

Clause 122.3A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:404; a CDR2 sequence consisting of SEQ ID NO:406; and a CDR3sequence consisting of SEQ ID NO:408, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:424; a CDR2 sequenceconsisting of SEQ ID NO:426; and a CDR3 sequence consisting of SEQ IDNO:428;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:402, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:422;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:402, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:422;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:401,and/or a light chain having the amino acid sequence of SEQ ID NO:421.

Clause 122.4A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:444; a CDR2 sequence consisting of SEQ ID NO:446; and a CDR3sequence consisting of SEQ ID NO:448, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:464; a CDR2 sequenceconsisting of SEQ ID NO:466; and a CDR3 sequence consisting of SEQ IDNO:468;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:442, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:462;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:442, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:462;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:441,and/or a light chain having the amino acid sequence of SEQ ID NO:461.

Clause 122.5A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:484; a CDR2 sequence consisting of SEQ ID NO:486; and a CDR3sequence consisting of SEQ ID NO:488, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:504; a CDR2 sequenceconsisting of SEQ ID NO:506; and a CDR3 sequence consisting of SEQ IDNO:508;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:482, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:502;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:482, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:502;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:481,and/or a light chain having the amino acid sequence of SEQ ID NO:501.

Clause 122.6A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:524; a CDR2 sequence consisting of SEQ ID NO:526; and a CDR3sequence consisting of SEQ ID NO:528, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:544; a CDR2 sequenceconsisting of SEQ ID NO:546; and a CDR3 sequence consisting of SEQ IDNO:548;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:522, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:542;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:522, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:542;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:521,and/or a light chain having the amino acid sequence of SEQ ID NO:541.

Clause 122.7A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:564; a CDR2 sequence consisting of SEQ ID NO:566; and a CDR3sequence consisting of SEQ ID NO:568, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:584; a CDR2 sequenceconsisting of SEQ ID NO:586; and a CDR3 sequence consisting of SEQ IDNO:588;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:562, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:582;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:562, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:582;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:561,and/or a light chain having the amino acid sequence of SEQ ID NO:581.

Clause 122.8A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:604; a CDR2 sequence consisting of SEQ ID NO:606; and a CDR3sequence consisting of SEQ ID NO:608, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:624; a CDR2 sequenceconsisting of SEQ ID NO:626; and a CDR3 sequence consisting of SEQ IDNO:628;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:602, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:622;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:602, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:622;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:601,and/or a light chain having the amino acid sequence of SEQ ID NO:621.

Clause 122.9A. The method of any one of Clauses 98A-110A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragmentcomprises:

(a) a variable heavy chain comprising a CDR1 sequence consisting of SEQID NO:644; a CDR2 sequence consisting of SEQ ID NO:646; and a CDR3sequence consisting of SEQ ID NO:648, and/or a variable light chaincomprising a CDR1 sequence consisting of SEQ ID NO:664; a CDR2 sequenceconsisting of SEQ ID NO:666; and a CDR3 sequence consisting of SEQ IDNO:668;

(b) a variable heavy chain comprising an amino acid sequence with atleast 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ IDNO:642, and/or a variable light chain comprising an amino acid sequencewith at least 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity toSEQ ID NO:662;

(c) a variable heavy chain having the amino acid sequence of SEQ IDNO:642, and/or a variable light chain having the amino acid sequence ofSEQ ID NO:662;

(d) a heavy chain having the amino acid sequence of SEQ ID NO:641,and/or a light chain having the amino acid sequence of SEQ ID NO:661.

Clause 123A. The method of any one of Clauses 98A-122.9A, wherein the atleast one anti-human ACTH antibody or antibody fragment is selected fromthe group consisting of chimeric, humanized, and human antibodies orantibody fragments.

Clause 124A. The method of any one of Clauses 98A-123A, wherein the atleast one anti-human ACTH antibody or antibody fragment is selected fromthe group consisting of scFvs, camelbodies, nanobodies, IgNAR, Fabfragments, Fab′ fragments, MetMab like antibodies, monovalent antibodyfragments, and F(ab′)₂ fragments.

Clause 125A. The method of any one of Clauses 98A-124A, wherein the atleast one anti-human ACTH antibody or antibody fragment substantially orentirely lacks N-glycosylation and/or O-glycosylation.

Clause 126A. The method of any one of Clauses 98A-125A, wherein the atleast one anti-human ACTH antibody or antibody fragment comprises ahuman constant domain.

Clause 127A. The method of any one of Clauses 98A-126A, wherein the atleast one anti-human ACTH antibody or antibody fragment is an IgG1,IgG2, IgG3, or IgG4 antibody.

Clause 128A. The method of any one of Clauses 98A-127A, wherein the atleast one anti-human ACTH antibody or antibody fragment comprises an Fcregion that has been modified to alter at least one of effectorfunction, half-life, proteolysis, or glycosylation.

Clause 129A. The method of Clause 128A, wherein the Fc region containsone or more mutations that alters or eliminates N- and/orO-glycosylation.

Clause 130A. The method of any one of Clauses 98A-129A, wherein the atleast one anti-human ACTH antibody or antibody fragment is a humanizedantibody or antibody fragment.

Clause 131A. The method of any one of Clauses 98A-130A, wherein the atleast one anti-human ACTH antibody or antibody fragment binds to ACTHwith a K_(D) of less than or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M.

Clause 132A. The method of any one of Clauses 98A-131A, wherein the atleast one anti-human ACTH antibody or antibody fragment binds to ACTHwith a K_(D) of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, or 10⁻¹² M.

Clause 133A. The method of any one of Clauses 98A-132A, wherein the atleast one anti-human ACTH antibody or antibody fragment binds to ACTHwith an off-rate (k_(d)) of less than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹,5×10⁻⁵ s⁻¹, or 10⁻⁵ s⁻¹.

Clause 134A. The method of any one of Clauses 98A-133A, wherein the atleast one anti-human ACTH antibody or antibody fragment is directly orindirectly attached to a therapeutic agent.

Clause 135A. The method of any one of Clauses 98A-134A, wherein the atleast one anti-human ACTH antibody or antibody fragment is attached toone or more detectable moieties.

Clause 136A. The method of Clause 135A, wherein detectable moietycomprises a fluorescent dye, enzyme, substrate, bioluminescent material,radioactive material, chemiluminescent moiety, or mixtures thereof.

Clause 137A. The method of any one of Clauses 98A-136A, wherein the atleast one anti-human ACTH antibody or antibody fragment is attached toone or more functional moieties.

Clause 138A. The method of any one of Clauses 98A-137A, wherein the atleast one isolated anti-human ACTH antibody or antibody fragment reducesplasma cortisol, corticosterone and/or aldosterone levels.

Clause 139A. The method of any one of Clauses 98A-138A, wherein themethod further comprises administering separately or co-administeringanother agent.

Clause 140A. The method of Clause 139A, wherein (i) the other agent isselected from the group consisting of ketoconazole (Nizoral®),aminoglutethimide (Cytadren®), metyrapone (Metopirone®), mitotane(Lysodren®) etomidate (Amidate®), cyproheptadine (Periactin® orPeritol®), valproic acid (Depakote®), cabergoline (Dostinex®),pasireotide (Signifor®), rosiglitazone (Avandia®), conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), and satavaptan(SR121463, planned trade name Aquilda®), mifepristone (Korlym®),armodafinil (Nuvigil®) and modafinil (Provigil®) or wherein the otheractive agent is selected from the group consisting of: Accupril(quinapril), Aceon (perindopril), Adalat, Adalat CC, Aldactone(spironolactone), aldosterone receptor blockers, alpha-adrenergicreceptor blockers, alpha-glucosidase inhibitors, Altace (ramipril),Alteplase, aminoglutethimide (Cytadren®), amiodarone, angiotensinconverting enzyme (ACE) Inhibitors, angiotensin II receptor antagonists,Angiotensin II receptor blockers (ARBs), antiarrhythmics,anti-cholesterol drugs, anti-clotting agents, antidiabetogenic drugs,anti-hypertensive agents, antiplatelet drugs, ApoA-1 mimics, aspirin,Atacand (candesartan), Avapro (irbesartan), beta blockers,beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR, CETPinhibitors, conivaptan (Vaprisol®), Cordarone (amiodarone), Coreg(carvedilol), Covera-HS, Cozaar (losartan), cyproheptadine (Periactin®or Peritol®), Demadex (torsemide), digoxin, Dilacor XR, Dilatrate-SR,Diltia XT, Diovan (valsartan), dipeptidyl peptidase-4 inhibitors,diuretics, Dobutrex (dobutamine), drugs that suppress ACTH secretion,drugs that suppress cortisol secretion, dual angiotensin convertingenzyme/neutral endopeptidase (ACE/NEP) inhibitors, endothelinantagonists, endothelin receptor blockers, Esidrix(hydrochlorothiazide), etomidate (Amidate®), Fragmin, gemfibrozil(Lopid, Gemcor), glucocorticoid receptor antagonists, heart failuredrugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine (Questran),IMDUR (isosorbide mononitrate), Inderal (propranolol), inhibitors of aNa—K-ATPase membrane pump, inhibitors of steroidogenesis, insulintherapies, Iso-Bid, Isonate, Isoptin, Isoptin SR, Isorbid (isosorbidedinitrate), Isordil, Isotrate, ketoconazole (Nizoral®), Lasix(furosemide), lixivaptan (VPA-985), Lopressor, Lotensin (benazepril),Lovenox, Mavik (trandolapril), meglitinides, metyrapone (Metopirone®),Micardis (telmisartan), mifepristone (Korlym®), mitotane (Lysodren®),Monopril (fosinopril), neutral endopeptidase (NEP) inhibitors,Normodyne, Norvasc (amlodipine), obesity-reducing agents, Omacor,pantethine, pasireotide (Signifor®), Plendil (felodipine), PPAR-gammaagonists, Primacor (milrinone), Prinivil, Procanbid (procainamide),Procardia, Procardia XL (nifedipine), renin inhibitors, Reteplase,rosiglitazone (Avandia®), satavaptan (SR121463, planned trade nameAquilda®), Sectral (acebutolol), somatostatin analogs, Sorbitrate(isosorbide dinitrate), statins, Streptokinase, Sular (nisoldipine),sulfonylurea, Tambocor (flecainide), Tenecteplase, Tenormin (atenolol),thiazolidinediones, Tiazac (diltiazem), Tissue plasminogen activator(TPA), tolvaptan (OPC-41061), Toprol-XL (metoprolol), Trandate(labetalol), Univasc (moexipril), Urokinase, valproic acid (Depakote®),vaptans, Vascor (bepridil), vasodilators, Vasodilators, vasopressinantagonists, Vasotec (enalapril), Verelan, Verelan PM (verapamil),warfarin (Coumadin), Zaroxolyn (metolazone), Zebeta (bisoprolol), andZestril (lisinopril), and/or (ii) the other active agent is selectedfrom the group consisting of: corticosteroids, glucocorticoids,mineralocorticoids, cortisol (hydrocortisone), dexamethasone, cortisone,prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone,fludrocortisone acetate, deoxycorticosterone, deoxycorticosteroneacetate (DOCA), and aldosterone.

Clause 141A. The method of Clause 139A or 140A, wherein the antibody orantibody fragment or the composition containing the antibody of antibodyfragment and the at least one other agent are administered concurrently.

Clause 142A. The method of Clause 139A or 140A, wherein the antibody orantibody fragment is administered before or after the at least one otheragent.

Clause 143A. The method of any one of Clauses 98A-138A, wherein themethod further comprises one or more of supplemental oxygen, continuouspositive airway pressure (CPAP), bilevel positive airway pressure(BPAP), expiratory positive airway pressure (EPAP), adaptiveservo-ventilation (ASV), oral applicanes, uvulopalatopharyngoplasty(UPPP), maxillomandibular advancement, nasal surgery, and removal oftonsils and/or adenoids.

Clause 1B. A human, humanized or chimerized anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment.

Clause 2B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment according to Clause 1B, which substantially does notinteract with (bind) a polypeptide consisting of: (i) the 13 N-terminalamino acid residues of ACTH (ACTH₁₋₁₃) and/or alpha-MSH, or (ii) the 22C-terminal amino acid residues of ACTH (ACTH) (Corticotrophin-LikeIntermediate Peptide or “CLIP”).

Clause 3B. The human, humanized or chimerized anti-ACTH antibody orantibody fragment according to Clause 1B, which binds to ACTH₁₋₃₉ with abinding affinity (K_(D)) at least 10-fold, 100-fold, 1000-fold or10,000-fold stronger than the binding affinity of said antibody orantibody fragment to (i) ACTH₁₋₁₃ and/or alpha-MSH, and/or (ii) CLIP(i.e., a numerically lower K_(D) for ACTH₁₋₃₉ by at least 10-fold,100-fold, 1000-fold or 10,000-fold relative to the K_(D) for ACTH₁₋₁₃and/or alpha-MSH and/or CLIP).

Clause 4B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-3B, which is a humanizedantibody or humanized antibody fragment.

Clause 5B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-3B, which is a human antibodyor human antibody fragment.

Clause 6B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-5B, which is selected fromthe group consisting of scFvs, camelbodies, nanobodies, IgNAR, Fabfragments, Fab′ fragments, MetMab like antibodies, monovalent antibodyfragments, and F(ab′)₂ fragments.

Clause 7B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-6B, which substantially orentirely lacks N-glycosylation and/or O-glycosylation.

Clause 8B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-7B, which comprises a humanconstant domain.

Clause 9B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of Clause 8B, which is an IgG1, IgG2, IgG3, or IgG4antibody.

Clause 10B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-9B, which comprises an Fcregion that has been modified to alter at least one of effectorfunction, half-life, proteolysis, or glycosylation.

Clause 11B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of Clause 10B, wherein the Fc region contains one ormore mutations that alters or eliminates N- and/or O-glycosylation.

Clause 12B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-11B, which binds to ACTH witha K_(D) of less than or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M,5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M.

Clause 13B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-12B, which binds to ACTH witha K_(D) of less than or equal to 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, or 10⁻¹² M.

Clause 14B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-13B, which binds to ACTH withan off-rate (kd) of less than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹, 5×10⁻⁵s⁻¹, or 10⁻⁵ s⁻¹.

Clause 15B. A human, humanized or chimerized anti-ACTH antibody orantibody fragment of any one of Clauses 1B-14B, which binds to ACTH witha K_(D) of less than about 100 nM, less than about 10 nM, less thanabout 1 nM, less than about 100 pM, less than about 50 pM, less thanabout 40 pM, less than about 25 pM, less than about 1 pM, between about10 pM and about 100 pM, between about 1 pM and about 100 pM, or betweenabout 1 pM and about 10 pM.

Clause 16B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-15B, wherein the antibody or antibody fragment is directlyor indirectly attached to a detectable label or therapeutic agent.

Clause 17B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-16B, which when administered to a human subject inhibitsor neutralizes at least one biological effect elicited by ACTH.

Clause 18B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-17B, which neutralizes or inhibits ACTH activation ofMC2R.

Clause 19B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-17B, which neutralizes or inhibits ACTH activation of atleast one of MC2R, MC3R, MC4R and MC5R.

Clause 20B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-17B, which neutralizes or inhibits ACTH activation of eachof MC2R, MC3R and MC4R.

Clause 21B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-17B, which inhibits ACTH-induced corticosterone secretion.

Clause 22B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-17B, which when administered to a human subject reducesplasma cortisol, corticosterone and/or aldosterone levels.

Clause 23B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-22B, wherein the antibody or antibody fragment is capableof inhibiting the binding of ACTH to a MCR.

Clause 24B. The anti-human ACTH antibody or antibody fragment of Clause23B, wherein the MCR is at least one of MC1R, MC2R, MC3R, MC4R and MC5R;at least one of MC2R, MC3R, and MC4R; each of MC2R, MC3R, and MC4R; oreach of MC1R, MC2R, MC3R, MC4R and MC5R.

Clause 25B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-24B, wherein the antibody or antibody fragment binds toACTH with a K_(D) that is less than about 100 nM.

Clause 26B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-24B, which binds to ACTH with a K_(D) that is less thanabout 100 pM.

Clause 27B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-24B, which binds to ACTH with a K_(D) that is less thanabout 50 pM.

Clause 28B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-24B, which binds to ACTH with a K_(D) that is less thanabout 25 pM.

Clause 29B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-24B, which binds to ACTH with a K_(D) that is betweenabout 10 pM and about 100 pM.

Clause 30B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-24B, which binds to ACTH with a K_(D) that is less thanabout 40 nM.

Clause 31B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-30B, which has stronger affinity for ACTH₁₋₃₉ as comparedto alpha-MSH or CLIP and/or does not bind to alpha-MSH or CLIP.

Clause 32B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-31B, wherein the antibody or antibody fragment is attachedto at least one effector moiety.

Clause 33B. The anti-human ACTH antibody or antibody fragment of Clause32B, wherein effector moiety comprises a chemical linker.

Clause 34B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-33B, wherein the antibody or antibody fragment is attachedto one or more detectable moieties.

Clause 35B. The anti-human ACTH antibody or antibody fragment of Clause34B, wherein detectable moiety comprises a fluorescent dye, enzyme,substrate, bioluminescent material, radioactive material,chemiluminescent moiety, or mixtures thereof.

Clause 36B. The anti-human ACTH antibody or antibody fragment of any oneof Clauses 1B-35B, wherein the antibody or antibody fragment is attachedto one or more functional moieties.

Clause 37B. An antibody produced against an anti-human ACTH antibody oranti-ACTH antibody fragment according to any one of Clauses 1B-36B.

Clause 38B. The antibody of Clause 37B, which is an anti-idiotypicantibody.

Clause 39B. A method of using an anti-idiotypic antibody or antibodyfragment according to Clause 38B to detect the levels of said anti-ACTHantibody or antibody fragment and/or to neutralize said anti-ACTHantibody or antibody fragment in a subject administered said anti-ACTHantibody or antibody fragment.

Clause 40B. A composition suitable for therapeutic, prophylactic, or adiagnostic use comprising a therapeutically, prophylactically ordiagnostically effective amount of at least one anti-human ACTH antibodyor antibody fragment according to any one of Clauses 1B-39B.

Clause 41B. The composition of Clause 39B, which is suitable forsubcutaneous administration.

Clause 42B. The composition of Clause 39B, which is suitable forintravenous administration.

Clause 43B. The composition of Clause 39B, which is lyophilized.

Clause 44B. The composition of any one of Clauses 39B-43B, furthercomprising a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative, or mixture thereof.

Clause 45B. The composition of any one of Clauses 39B-44B, furthercomprising another active agent.

Clause 46B. The composition of Clause 45B, wherein (i) the other activeagent is selected from the group consisting of ketoconazole (Nizoral®),aminoglutethimide (Cytadren®), metyrapone (Metopirone®), mitotane(Lysodren®) etomidate (Amidate®), cyproheptadine (Periactin® orPeritol®), valproic acid (Depakote®), cabergoline (Dostinex®),pasireotide (Signifor®), rosiglitazone (Avandia®), conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), satavaptan(SR121463, planned trade name Aquilda®), mifepristone (Korlym®),armodafinil (Nuvigil®) and modafinil (Provigil®) or wherein the otheractive agent is selected from the group consisting of: Accupril(quinapril), Aceon (perindopril), Adalat, Adalat CC, Aldactone(spironolactone), aldosterone receptor blockers, alpha-adrenergicreceptor blockers, alpha-glucosidase inhibitors, Altace (ramipril),Alteplase, aminoglutethimide (Cytadren®), amiodarone, angiotensinconverting enzyme (ACE) Inhibitors, angiotensin II receptor antagonists,Angiotensin II receptor blockers (ARBs), antiarrhythmics,anti-cholesterol drugs, anti-clotting agents, antidiabetogenic drugs,anti-hypertensive agents, antiplatelet drugs, ApoA-1 mimics, aspirin,Atacand (candesartan), Avapro (irbesartan), beta blockers,beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR, CETPinhibitors, conivaptan (Vaprisol®), Cordarone (amiodarone), Coreg(carvedilol), Covera-HS, Cozaar (losartan), cyproheptadine (Periactin®or Peritol®), Demadex (torsemide), digoxin, Dilacor XR, Dilatrate-SR,Diltia XT, Diovan (valsartan), dipeptidyl peptidase-4 inhibitors,diuretics, Dobutrex (dobutamine), drugs that suppress ACTH secretion,drugs that suppress cortisol secretion, dual angiotensin convertingenzyme/neutral endopeptidase (ACE/NEP) inhibitors, endothelinantagonists, endothelin receptor blockers, Esidrix(hydrochlorothiazide), etomidate (Amidate®), Fragmin, gemfibrozil(Lopid, Gemcor), glucocorticoid receptor antagonists, heart failuredrugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine (Questran),IMDUR (isosorbide mononitrate), Inderal (propranolol), inhibitors of aNa—K-ATPase membrane pump, inhibitors of steroidogenesis, insulintherapies, Iso-Bid, Isonate, Isoptin, Isoptin SR, Isorbid (isosorbidedinitrate), Isordil, Isotrate, ketoconazole (Nizoral®), Lasix(furosemide), lixivaptan (VPA-985), Lopressor, Lotensin (benazepril),Lovenox, Mavik (trandolapril), meglitinides, metyrapone (Metopirone®),Micardis (telmisartan), mifepristone (Korlym®), mitotane (Lysodren®),Monopril (fosinopril), neutral endopeptidase (NEP) inhibitors,Normodyne, Norvasc (amlodipine), obesity-reducing agents, Omacor,pantethine, pasireotide (Signifor®), Plendil (felodipine), PPAR-gammaagonists, Primacor (milrinone), Prinivil, Procanbid (procainamide),Procardia, Procardia XL (nifedipine), renin inhibitors, Reteplase,rosiglitazone (Avandia®), satavaptan (SR121463, planned trade nameAquilda®), Sectral (acebutolol), somatostatin analogs, Sorbitrate(isosorbide dinitrate), statins, Streptokinase, Sular (nisoldipine),sulfonylurea, Tambocor (flecainide), Tenecteplase, Tenormin (atenolol),thiazolidinediones, Tiazac (diltiazem), Tissue plasminogen activator(TPA), tolvaptan (OPC-41061), Toprol-XL (metoprolol), Trandate(labetalol), Univasc (moexipril), Urokinase, valproic acid (Depakote®),vaptans, Vascor (bepridil), vasodilators, Vasodilators, vasopressinantagonists, Vasotec (enalapril), Verelan, Verelan PM (verapamil),warfarin (Coumadin), Zaroxolyn (metolazone), Zebeta (bisoprolol), andZestril (lisinopril), and/or (ii) the other active agent is selectedfrom the group consisting of: corticosteroids, glucocorticoids,mineralocorticoids, cortisol (hydrocortisone), dexamethasone, cortisone,prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone,fludrocortisone acetate, deoxycorticosterone, deoxycorticosteroneacetate (DOCA), and aldosterone.

Clause 47B. The composition of any one of Clauses 39B-46B, which islyophilized, stabilized and/or formulated for administration byinjection.

Clause 48B. An isolated nucleic acid sequence or nucleic acid sequencesencoding an anti-human ACTH antibody or antibody fragment oranti-idiotypic antibody or antibody fragment according to any one ofClauses 1B-37B.

Clause 49B. A vector or vectors containing the isolated nucleic acidsequence or sequences of Clause 48B.

Clause 50B. A host cell comprising the isolated nucleic acid sequence orsequences of Clause 46B or the vector or vectors of Clause 49B.

Clause 51B. The host cell of Clause 50B, which is a mammalian,bacterial, fungal, yeast, avian or insect cell.

Clause 52B. The host cell of Clause 51B, which is a filamentous fungi ora yeast.

Clause 53B. The host cell of Clause 52B, wherein the yeast is selectedfrom the from the following genera: Arxiozyma; Ascobotryozyma;Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia;Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia;Saccharomyces; Saturnispora; Tetrapisispora; Torulaspora; Williopsis;and Zygosaccharomyces.

Clause 54B. The host cell of Clause 53B, which is the yeast genus isPichia.

Clause 55B. The host cell of Clause 54B, wherein the species of Pichiais selected from Pichia pastoris, Pichia methanolica and Hansenulapolymorpha (Pichia angusta).

Clause 56B. A method of making an anti-human ACTH antibody or antibodyfragment comprising culturing the host cell of any one of Clauses50B-55B under conditions that provide for expression of said antibody orantibody fragment.

Clause 57B. The method of Clause 56B, wherein the host cell is apolyploid yeast culture that stably expresses and secretes into theculture medium at least 10-25 mg/liter of said antibody or antibodyfragment.

Clause 58B. The method of Clause 57B, wherein said polyploidal yeast ismade by a method that comprises:

(i) introducing at least one expression vector containing one or moreheterologous polynucleotides encoding said antibody operably linked to apromoter and a signal sequence into a haploid yeast cell;

(ii) producing by mating or spheroplast fusion a polyploidal yeast fromsaid first and/or second haploid yeast cell;

(iii) selecting polyploidal yeast cells that stably express saidantibody; and

(iv) producing stable polyploidal yeast cultures from said polyploidalyeast cells that stably express said antibody into the culture medium.

Clause 59B. The method of Clause 58B, wherein said yeast is of the genusPichia.

Clause 60B. A method for blocking, inhibiting or neutralizing one ormore biological effects associated with ACTH comprising administering toa subject in need thereof an effective amount of an anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment.

Clause 61B. A method for treating or preventing a condition associatedwith elevated ACTH levels in a subject, comprising administering to asubject in need thereof an effective amount of an anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment.

Clause 62B. A method for treating or preventing a condition associatedwith elevated cortisol, corticosterone and/or aldosterone levels in asubject, comprising administering to the subject in need thereof aneffective amount of an anti-human adrenocorticotrophic hormone (“ACTH”)antibody or antibody fragment.

Clause 63B. The method of any one of Clauses 60B-62B, wherein (i) thecondition is selected from the group consisting of ACTH-drivenhypercortisolism (Cushing's Disease and/or Cushing's Syndrome), obesity,diabetes, Parkinson's disease, sleep disorders including e.g., insomnia,sleep apnea, narcolepsy, depression, anxiety disorders, cancer (such asCushing's Syndrome resulting from ectopic ACTH expression, e.g., insmall cell lung cancer, non-small cell lung cancer (NSCLC), pancreaticcarcinoma, neural tumors, or thymoma), muscle atrophies, hypertension,Alzheimer's disease, dementia and other cognitive dysfunction disorders,Alzheimer's disease, galactorrhea, stress related disorders, heartfailure, diabetes, hyperinsulinemia, metabolic syndromes,hyperaldosteronism, Conn's syndrome and familial hyperaldosteronism, or(ii) wherein the condition comprises congenital adrenal hyperplasia(CAH), Classical CAH, or Nonclassical CAH, or (iii) wherein thecondition comprises familial glucocorticoid deficiency (FGD) or (iv)wherein the condition comprises Allgrove syndrome, bilateraladrenalectomy, or Nelson's syndrome.

Clause 64B. A method for neutralizing ACTH-induced MCR signaling,comprising administering to a subject in need thereof an effectiveamount of an anti-human adrenocorticotrophic hormone (“ACTH”) antibodyor antibody fragment.

Clause 65B. A method for inhibiting ACTH-induced cortisol,corticosterone and/or aldosterone secretion, comprising administering toa subject in need thereof an effective amount of an anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment.

Clause 66B. A method for reducing ACTH-induced plasma cortisol,corticosterone and/or aldosterone levels in a subject in need thereof,comprising administering to the subject in need thereof an effectiveamount of a human, humanized or chimerized anti-humanadrenocorticotrophic hormone (“ACTH”) antibody or antibody fragment.

Clause 67B. The method of any one of Clauses 60B-66B, wherein theantibody is a human, humanized or chimerized anti-ACTH antibody orantibody fragment

Clause 68B. The method of any one of Clauses 60B-67B, wherein theantibody or antibody fragment substantially does not interact with(bind) a polypeptide consisting of: (i) the 13 N-terminal amino acidresidues of ACTH (ACTH₁₋₁₃) and/or alpha-MSH, or (ii) the 22 C-terminalamino acid residues of ACTH (ACTH₁₈₋₃₉).

Clause 69B. The method of any one of Clauses 60B-68B, wherein the atleast one anti-human ACTH antibody or antibody fragment is selected fromthe group consisting of scFvs, camelbodies, nanobodies, IgNAR, Fabfragments, Fab′ fragments, MetMab like antibodies, monovalent antibodyfragments, and F(ab′)₂ fragments.

Clause 70B. The method of any one of Clauses 60B-69B, wherein the atleast one anti-human ACTH antibody or antibody fragment substantially orentirely lacks N-glycosylation and/or O-glycosylation.

Clause 71B. The method of any one of Clauses 60B-70B, wherein the atleast one anti-human ACTH antibody or antibody fragment comprises ahuman constant domain.

Clause 72B. The method of any one of Clauses 60B-71B, wherein the atleast one anti-human ACTH antibody or antibody fragment is an IgG1,IgG2, IgG3, or IgG4 antibody.

Clause 73B. The method of any one of Clauses 60B-72B, wherein the atleast one anti-human ACTH antibody or antibody fragment comprises an Fcregion that has been modified to alter at least one of effectorfunction, half-life, proteolysis, or glycosylation.

Clause 74B. The method of Clause 73B, wherein the Fc region contains oneor more mutations that alters or eliminates N- and/or O-glycosylation.

Clause 75B. The method of any one of Clauses 60B-74B, wherein the atleast one anti-human ACTH antibody or antibody fragment is a humanizedantibody or antibody fragment.

Clause 76B. The method of any one of Clauses 60B-75B, wherein the atleast one anti-human ACTH antibody or antibody fragment binds to ACTHwith a K_(D) of less than or equal to 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, or 10⁻¹³ M.

Clause 77B. The method of any one of Clauses 60B-76B, wherein the atleast one anti-human ACTH antibody or antibody fragment binds to ACTHwith a K_(D) of less than or equal to 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, or 10⁻¹² M.

Clause 78B. The method of any one of Clauses 60B-77B, wherein the atleast one anti-human ACTH antibody or antibody fragment binds to ACTHwith an off-rate (kd) of less than or equal to 5×10⁻⁴ s⁻¹, 10⁻⁴ s⁻¹,5×10⁵ s⁻¹, or 10⁻⁵ s⁻¹.

Clause 79B. The method of any one of Clauses 60B-78B, wherein the atleast one anti-human ACTH antibody or antibody fragment is directly orindirectly attached to a therapeutic agent.

Clause 80B. The method of any one of Clauses 60B-79B, wherein the atleast one anti-human ACTH antibody or antibody fragment is attached toone or more detectable moieties.

Clause 81B. The method of Clause 80B, wherein detectable moietycomprises a fluorescent dye, enzyme, substrate, bioluminescent material,radioactive material, chemiluminescent moiety, or mixtures thereof.

Clause 82B. The method of any one of Clauses 60B-81B, wherein the atleast one anti-human ACTH antibody or antibody fragment is attached toone or more functional moieties.

Clause 83B. The method of any one of Clauses 60B-82B, wherein the atleast one isolated anti-human ACTH antibody or antibody fragment reducesplasma cortisol, corticosterone and/or aldosterone levels.

Clause 84B. The method of any one of Clauses 60B-83B, wherein the methodfurther comprises administering separately or co-administering anotheragent.

Clause 85B. The method of Clause 84B, or wherein the other agent isselected from the group consisting of (i) ketoconazole (Nizoral®),aminoglutethimide (Cytadren®), metyrapone (Metopirone®), mitotane(Lysodren®) etomidate (Amidate®), cyproheptadine (Periactin® orPeritol®), valproic acid (Depakote®), cabergoline (Dostinex®),pasireotide (Signifor®), rosiglitazone (Avandia®), conivaptan(Vaprisol®), tolvaptan (OPC-41061), lixivaptan (VPA-985), satavaptan(SR121463, planned trade name Aquilda®), mifepristone (Korlym®),armodafinil (Nuvigil®) and modafinil (Provigil®) or wherein the otheractive agent is selected from the group consisting of: Accupril(quinapril), Aceon (perindopril), Adalat, Adalat CC, Aldactone(spironolactone), aldosterone receptor blockers, alpha-adrenergicreceptor blockers, alpha-glucosidase inhibitors, Altace (ramipril),Alteplase, aminoglutethimide (Cytadren®), amiodarone, angiotensinconverting enzyme (ACE) Inhibitors, angiotensin II receptor antagonists,Angiotensin II receptor blockers (ARBs), antiarrhythmics,anti-cholesterol drugs, anti-clotting agents, antidiabetogenic drugs,anti-hypertensive agents, antiplatelet drugs, ApoA-1 mimics, aspirin,Atacand (candesartan), Avapro (irbesartan), beta blockers,beta-adrenergic receptor blockers, Betapace (sotalol), BiDil(hydralazine with isosorbide dinitrate), biguanides, blood thinners,Brevibloc (esmolol), Bumex (bumetanide), cabergoline (Dostinex®), Caduet(a combination of a statin cholesterol drug and amlodipine), Calan,Calan SR, Calcium channel blockers, Capoten (captopril), Cardene,Cardene SR (nicardipine), Cardizem, Cardizem CD, Cardizem SR, CETPinhibitors, conivaptan (Vaprisol®), Cordarone (amiodarone), Coreg(carvedilol), Covera-HS, Cozaar (losartan), cyproheptadine (Periactin®or Peritol®), Demadex (torsemide), digoxin, Dilacor XR, Dilatrate-SR,Diltia XT, Diovan (valsartan), dipeptidyl peptidase-4 inhibitors,diuretics, Dobutrex (dobutamine), drugs that suppress ACTH secretion,drugs that suppress cortisol secretion, dual angiotensin convertingenzyme/neutral endopeptidase (ACE/NEP) inhibitors, endothelinantagonists, endothelin receptor blockers, Esidrix(hydrochlorothiazide), etomidate (Amidate®), Fragmin, gemfibrozil(Lopid, Gemcor), glucocorticoid receptor antagonists, heart failuredrugs, Heparin, HMG-Co-A reductase inhibitors, holestyramine (Questran),IMDUR (isosorbide mononitrate), Inderal (propranolol), inhibitors of aNa—K-ATPase membrane pump, inhibitors of steroidogenesis, insulintherapies, Iso-Bid, Isonate, Isoptin, Isoptin SR, Isorbid (isosorbidedinitrate), Isordil, Isotrate, ketoconazole (Nizoral®), Lasix(furosemide), lixivaptan (VPA-985), Lopressor, Lotensin (benazepril),Lovenox, Mavik (trandolapril), meglitinides, metyrapone (Metopirone®),Micardis (telmisartan), mifepristone (Korlym®), mitotane (Lysodren®),Monopril (fosinopril), neutral endopeptidase (NEP) inhibitors,Normodyne, Norvasc (amlodipine), obesity-reducing agents, Omacor,pantethine, pasireotide (Signifor®), Plendil (felodipine), PPAR-gammaagonists, Primacor (milrinone), Prinivil, Procanbid (procainamide),Procardia, Procardia XL (nifedipine), renin inhibitors, Reteplase,rosiglitazone (Avandia®), satavaptan (SR121463, planned trade nameAquilda®), Sectral (acebutolol), somatostatin analogs, Sorbitrate(isosorbide dinitrate), statins, Streptokinase, Sular (nisoldipine),sulfonylurea, Tambocor (flecainide), Tenecteplase, Tenormin (atenolol),thiazolidinediones, Tiazac (diltiazem), Tissue plasminogen activator(TPA), tolvaptan (OPC-41061), Toprol-XL (metoprolol), Trandate(labetalol), Univasc (moexipril), Urokinase, valproic acid (Depakote®),vaptans, Vascor (bepridil), vasodilators, Vasodilators, vasopressinantagonists, Vasotec (enalapril), Verelan, Verelan PM (verapamil),warfarin (Coumadin), Zaroxolyn (metolazone), Zebeta (bisoprolol), andZestril (lisinopril), and/or (ii) the other active agent is selectedfrom the group consisting of: corticosteroids, glucocorticoids,mineralocorticoids, cortisol (hydrocortisone), dexamethasone, cortisone,prednisone, prednisolone, methylprednisolone, dexamethasone,betamethasone, triamcinolone, beclometasone, fludrocortisone,fludrocortisone acetate, deoxycorticosterone, deoxycorticosteroneacetate (DOCA), and aldosterone.

Clause 86B. The method of Clause 84B or 85B, wherein the antibody orantibody fragment or the composition containing the antibody of antibodyfragment and the at least one other agent are administered concurrently.

Clause 87B. The method of Clause 84B or 85B, wherein the antibody orantibody fragment is administered before or after the at least one otheragent.

Clause 88B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-ACTH antibody or antibody fragment which substantiallydoes not interact with (bind) a polypeptide consisting of: (i) the 13N-terminal amino acid residues of ACTH (ACTH₁₋₁₃) and/or alpha-MSH, or(ii) the 22 C-terminal amino acid residues of ACTH (ACTH₁₈₋₃₉)(Corticotrophin-Like Intermediate peptide or “CLIP”).

Clause 89B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-ACTH antibody or antibody fragment which binds toACTH₁₋₃₉ with a binding affinity (K_(D)) at least 10-fold, 100-fold,1000-fold or 10,000-fold stronger than the binding affinity of saidantibody or antibody fragment to (i) ACTH₁₋₁₃ and/or alpha-MSH, and/or(ii) CLIP (i.e., a numerically lower K_(D) for ACTH₁₋₃₉ than forACTH₁₋₁₃ and/or alpha-MSH and/or CLIP by at least 10-fold, 100-fold,1000-fold or 10,000-fold).

Clause 90B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-human ACTH antibody or antibody fragment whichneutralizes or inhibits ACTH activation of MC2R.

Clause 91B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-human ACTH antibody or antibody fragment whichneutralizes or inhibits ACTH activation of at least one of MC2R, MC3Rand MC4R.

Clause 92B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-human ACTH antibody or antibody fragment, whichneutralizes or inhibits ACTH activation of each of MC2R, MC3R and MC4R.

Clause 93B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-human ACTH antibody or antibody fragment, which inhibitsACTH-induced corticosterone secretion.

Clause 94B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-human ACTH antibody or antibody fragment, which whenadministered to a human subject reduces plasma cortisol, corticosteroneand/or aldosterone levels.

Clause 95B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-human ACTH antibody or antibody fragment capable ofinhibiting the binding of ACTH to a MCR.

Clause 96B. The method of any one of Clauses 60B-87B, wherein theanti-ACTH antibody or antibody fragment is a human, humanized orchimerized anti-human ACTH antibody or antibody fragment, capable ofinhibiting the binding of ACTH to at least one of MC1R, MC2R, MC3R, MC4Rand MC5R; at least one of MC2R, MC3R, and MC4R; each of MC2R, MC3R, andMC4R; or each of MC1R, MC2R, MC3R, MC4R and MC5R.

Clause 97B. The method of any one of Clauses 60B-83B, wherein the methodfurther comprises one or more of supplemental oxygen, continuouspositive airway pressure (CPAP), bilevel positive airway pressure(BPAP), expiratory positive airway pressure (EPAP), adaptiveservo-ventilation (ASV), oral applicanes, uvulopalatopharyngoplasty(UPPP), maxillomandibular advancement, nasal surgery, and removal oftonsils and/or adenoids.

The entire disclosure of each document cited herein (including patents,patent applications, journal articles, abstracts, manuals, books, orother disclosures) including all references cited herein (including,without limitation thereto, in the Background, Detailed Description, andExamples) is hereby incorporated by reference in its entirety.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXAMPLES Example 1 Preparation of Antibodies that Selectively Bind ACTH

By using an antibody selection protocol substantially as describedherein, a panel of antibodies specific to ACTH was produced.

Immunization Strategy

Rabbits were immunized with ACTH 1-24 (Bachem, Torrance, Calif.) (SEQ IDNO: 1122) or ACTH 1-39 (Bachem) (SEQ ID NO:1121). Peptides were preparedfor immunization as follows. A volume of 1 ml of 10 mg/ml KLH wasdissolved in DPBS supplemented to 1M NaCl and combined with 0.5 ml of 5mg/ml peptide (dissolved in deionized water). Then 1.4 ml of 40 mMCarbodiimide was added prior to a 12-hour incubation at room temperaturewith gentle mixing. Excess Carbodiimide and unconjugated peptide wereremoved by dialysis to DPBS prior to sterile filtration. Nextunconjugated peptide equal to the calculated mass of KLH was added tomake a final total protein concentration of 3.75 mg/ml.

Immunizations were performed by diluting 200 μg of antigen to 0.5 mlwith DPBS and mixing with an equal volume of complete Freund's adjuvantfor subcutaneous 1 ml injection at Day 1.

Boost injections of 100 ug were performed at Day 21, 42 and 60.

Antibody Selection Titer Assessment

To identify antibodies that neutralize ACTH 1-39 (SEQ ID NO:1121)induced signaling via MC2R, polyclonal antibody solutions were firstpurified via Protein A and dialyzed into a neutral buffer. Briefly,antibody solutions were incubated with ACTH 1-39 (SEQ ID NO:1121) at 3×the final concentration (100 pM) for 1 hr. While the antibody/antigencomplexes were incubated, MC2R expressing cells (Life Technologies,Grand Island, N.Y.) were detached with 0.25% trypsin for 4 minutes. Thecells were washed and re-suspended at 2×10⁶ cells per ml in assay buffer(Meso Scale Discovery [MSD], Rockville, Md.) and treated with 0.2 mMIBMX (Sigma, St. Louis Mo.). Ten microliters of cells was combined with20 μl of Ab/Ag mixture and added to a cAMP plate (MSD) and incubated for30 minutes at room temperature with shaking. Next 20 μl of labeled cAMPin cell lysis buffer (MSD) was added and incubated for 1 hour whileshaking. Following the incubation, 100 μl read buffer (MSD) was addedand read with a Sector Imager 2400.

Tissue Harvesting

Once acceptable titers were established, the rabbit(s) were sacrificed.Spleen, lymph nodes, and whole blood were harvested and processed asfollows:

Spleen and lymph nodes were processed into a single cell suspension bydisassociating the tissue and pushing through sterile wire mesh at 70 μm(Fisher) with a plunger of a 20 cc syringe. Cells were collected in PBS.Cells were washed twice by centrifugation. After the last wash, celldensity was determined by trypan blue. Cells were centrifuged at 1500RPM for 10 minutes; the supernatant was discarded. Cells wereresuspended in the appropriate volume of 10% dimethyl sulfoxide (DMSO,Sigma) in FBS (Hyclone) and dispensed at 1 ml/vial. Vials were stored at−70° C. in a slow freezing chamber for 24 hours and stored in liquidnitrogen.

Peripheral blood mononuclear cells (PBMCs) were isolated by mixing wholeblood with equal parts of PBS. 35 ml of the whole blood mixture wascarefully layered onto 8 ml of Lympholyte® Rabbit (Cedarlane,Burlington, Ontario, Canada) into a 45 ml conical tube (Corning) andcentrifuged 30 minutes at 2500 RPM at room temperature without brakes.After centrifugation, the PBMC layers were carefully removed using aglass Pasteur pipette (VWR), combined, and placed into a clean 50 mLvial. Cells were washed twice with PBS by centrifugation at 1500 RPM for10 minutes at room temperature, and cell density was determined bytrypan blue staining. After the last wash, cells were resuspended in anappropriate volume of 10% DMSO/FBS medium and frozen as described above.

B Cell Selection, Enrichment and Culture Conditions

On the day of setting up B cell culture, PBMC, splenocyte, or lymph nodevials were thawed for use. Vials were removed from LN2 tank and placedin a 37° C. water bath until thawed. Contents of vials were transferredinto 15 mL conical centrifuge tube (Corning) and 10 mL of modified RPMIwas slowly added to the tube. Cells were centrifuged for 5 minutes at 2KRPM, and the supernatant was discarded. Cells were resuspended in 10 mLof fresh media. Cell density and viability was determined by trypanblue.

For positive selection of anti-ACTH producing B-cells, biotinylatedhuman ACTH 1-39 (SEQ ID NO:1121) was pre-loaded onto the streptavidinbeads as follows. Seventy-five microliters of streptavidin beads(Miltenyi Biotec, Auburn Calif.) were mixed with N-terminallybiotinylated human ACTH 1-39 (1 μg/mL final concentration) and 300 μl ofPBS supplemented with 0.5% biotin free BSA and 2 mM EDTA (PBF). Thismixture was incubated at 4° C. for 30 minutes and unbound biotinylatedhuman ACTH 1-39 (Bachem) was removed using a MACS® separation column(Miltenyi Biotec) with a 1 ml rinse to remove unbound material. Thenbound material was plunged out by detachment from the magnet and used toresuspend cells from above in 100 μL per 1×10⁷ cells. The mixture wasthen incubated at 4° C. for 30 minutes and washed once with 10 mL ofPBF. After washing, the cells were resuspended in 500 μL of PBF and setaside. A MACS® MS column (Miltenyi Biotec) was pre-rinsed with 500 μL ofPBF on a magnetic stand (Miltenyi Biotec). Cell suspension was appliedto the column through a pre-filter, and unbound fraction was collected.The column was washed with 2.5 mL of PBF buffer. The column was removedfrom the magnet stand and placed onto a clean, sterile 1.5 mL Eppendorftube. 1 mL of PBF buffer was added to the top of the column, andpositive selected cells were collected. The yield and viability ofpositive cell fraction was determined by trypan blue staining. Positiveselection yielded an average of 1% of the starting cell concentration.

A pilot cell screen was established to provide information on seedinglevels for the culture. Plates were seeded at 5, 10, 25, 50, 100, or 200enriched B cells/well. In addition, each well contained 25-50Kcells/well of irradiated EL-4.B5 cells (5,000 Rads) and an appropriatelevel of activated rabbit T cell supernatant (See U.S. PatentApplication Publication No. 20070269868) (ranging from 1-5% depending onpreparation) in high glucose modified RPMI medium at a final volume of250 μL/well. Cultures were incubated for 5 to 7 days at 37° C. in 4%CO₂.

B-Cell Culture Screening by Antigen-Recognition (ELISA)

To identify wells producing anti-human ACTH antibodies, B-cellsupernatants were tested by antigen-recognition (ELISA). Briefly,neutravidin coated plates (Thermo Scientific), were coated with N-termbiotinylated human ACTH 1-39 (Bachem) (50 μl per well; 1 μg/ml) dilutedin ELISA buffer (0.5% fish skin gelatin in PBS pH 7.4) either forapproximately 1 hour at room temperature or alternatively overnight at4° C. The plates were then further blocked with ELISA buffer for onehour at room temperature and washed using wash buffer (PBS, 0.05% Tween20). B-cell supernatant samples (50 μL) were transferred onto the wellsand incubated for one hour at room temperature. After this incubation,the plate was washed with wash buffer. For development, an anti-rabbitspecific Fc-HRP (1:5000 dilution in ELISA buffer) was added onto thewells and incubated for 45 minutes at room temperature. After a 3× washstep with wash solution, the plate was developed using TMB substrate fortwo minutes at room temperature and the reaction was quenched using 0.5MHCl. The well absorbance was read at 450 nm.

To identify wells producing anti-human ACTH antibodies that do notrecognize ACTH 1-13 (SEQ ID NO: 1123) or ACTH 18-39 (SEQ ID NO: 1124),supernatant from wells positive for ACTH 1-39 binding by ELISA weretested by ELISA for binding to ACTH 1-13 and ACTH 18-39. Briefly, amixture of biotinylated ACTH 1-13 (SEQ ID NO:1121) and ACTH 18-39 (SEQID NO:1124) was bound onto Neutravidin coated plates (50 g per well, 1μg/μl each peptide). B-cell supernatant samples (50 μl) were testedwithout prior dilution. Recognition in this assay indicates crossreactivity with sub-peptide products of ACTH.

Identification of Functional Activity in B-Cell Supernatants Using Oneor More Assays

To identify wells producing anti-human ACTH antibodies that blocksignaling of ACTH via MC2R, supernatant from positive wells for ACTH1-39 binding by ELISA were tested in the cAMP assay (MSD) with MC2Rexpressing cells (Life Technologies). Supernatants (76 μl) werepre-incubated with 4 μl of a solution containing 3 nM ACTH 1-39 (Bachem)for 1 hour at room temperature. During the incubation, MC2R cells wereprepared as described for titer assessment. Cells (10 μl) andantigen/antibody complex (20 μl) were incubated together in a cAMP assayplate (MSD) and incubated at room temperature for 30 minutes whileshaking. Following the incubation, 20 μl of labeled cAMP in lysis buffer(MSD) was added and the plate was incubated for 1 hour while shaking.After the final incubation, 100 μl of 1.5× read buffer (MSD) was addedand plates read with a SECTOR® Imager 2400.

Alternatively, the supernatants were tested in a similar assay todetermine the ability to block signaling of ACTH in MC2R expressingcells via cAMP accumulation with a cAMP HTRF assay (Cisbio).Supernatants (78 μl) were pre-incubated 2 μl 5 nM ACTH 1-39 (Bachem) for1 hour at 37 C. During the incubation, MC2R cells were prepared asdescribed for titer assessment. Cells (10 μl) and antigen/antibodycomplex (40 μl) were transferred to an HTRF plate and shaken at roomtemperature for 30 minutes. Following the incubation, 20 μl of (1:20diluted) Eu3+ cryptate-labeled MAb anti-cAMP and 20 μl of (1:20 diluted)d2-labeled cAMP was added and the plate was incubated for 1 hour whileshaking. Following incubation plates were read (excitation 330, emission620/665 nM) and a ratio of 620:665 signal was determined.

Isolation of Antigen-Specific B Cells

Antigen-specific B cells were isolated (for general methods see co-ownedpublication no. WO/2014/146074, which is hereby incorporated byreference in its entirety). Plates containing wells of interest wereremoved from −70° C., and the cells from each well were recovered usingfive washes of 200 microliters of medium (10% RPMI complete, 55 μM BME)per well. The recovered cells were pelleted by centrifugation and thesupernatant was carefully removed. Cells from each well were thenre-suspended in 100 μl of medium and transferred to a 96 well plate.Cells were incubated for 90 minutes at 37° C. Following incubation,cells were pelleted by centrifugation, stained with a FITC-labeledanti-rabbit IgG (final concentration 6.25 μg/ml) (Creative Diagnostics,Shirley, N.Y.) and washed with up to 2 milliliters FACS buffer(Dulbecco's PBS w/2% FBS) and re-suspended in 250 ul of FACS buffer.

Control wells from the same culture sets that were similar incomposition to pooled wells of interest were thawed and stained alongside target wells. These samples were initially run on FACS (BD Influx)and gates were established for IgG, viability and physical parameters(FSC/SSC) that differentiate B cells from the murine EL4 cells. Oncegates were established, the sample of interest was run and IgG positive,viable cells that are of a consistent physical (FSC/SSC) population weresorted individually into wells of a 96 well plate pre-loaded with RT-PCRmaster mix. Upwards of 8 cells per well were sorted. Sorted plates wereremoved from the sorter and transferred directly to thermocyclers forPCR.

Amplification and Sequence Determination of Antibody Sequences fromFACS-Sorted B Cells

Antibody sequences were recovered using a combined RT-PCR based methodfrom a single cell sorted B-cell. Primers containing restriction enzymeswere designed to anneal in conserved and constant regions of the targetimmunoglobulin genes (heavy and light), such as rabbit immunoglobulinsequences, and a two-step nested PCR recovery was used to amplify theantibody sequence. Amplicons from each well were sequenced and analyzed.Representative antibodies from the resulting sequence clusters areselected for recombinant protein expression. The original heavy andlight variable regions amplified from rabbit cells are cloned into humanheavy and light chain constant region expression vectors via restrictionenzyme digestion and ligation. Vectors containing subcloned DNAfragments were amplified and purified. The sequences of the subclonedheavy and light chains were verified prior to expression.

Recombinant Production of Monoclonal Antibody of Desired AntigenSpecificity and/or Functional Properties

To determine antigen specificity and functional properties of recoveredantibodies from specific B-cells, the heavy and light chain plasmidswere co-transfected to generate rabbit/human chimeric antibodies fortesting. Briefly, heavy and light chimeric plasmids were transientlytransfected into HEK-293 cells. Transfections were allowed to incubatefor 5-7 days and upon harvest cells were pelleted by centrifugation.Supernatants were submitted for purification via Protein A. Resultingpurified chimeric antibodies were then evaluated in a variety of assaysto confirm specificity and potency.

Antigen-Recognition of Recombinant Antibodies by ELISA

To characterize recombinant expressed antibodies for their ability tobind to human ACTH 1-39 antibody-containing solutions were tested byELISA. All incubations were done at room temperature. Briefly, N-termbiotinylated human ACTH 1-39 was bound onto Neutravidin coated plates(Thermo Scientific) (50 μl per well, 1 μg/mL) in PBS) for 2 hours.ACTH-coated plates were then washed three times in wash buffer (PBS,0.05% Tween-20). The plates were then blocked using a blocking solution(PBS, 0.5% fish skin gelatin, 0.05% Tween-20) for approximately onehour. The blocking solution was then removed and the plates were thenincubated with a dilution series of the antibody being tested forapproximately one hour. At the end of this incubation, the plate waswashed three times with wash buffer and further incubated with asecondary antibody containing solution (Peroxidase conjugatedAffiniPure™ F(ab′)₂ fragment goat anti-human IgG, Fc fragment specific(Jackson Immunoresearch) for approximately 45 minutes and washed threetimes. Next a substrate solution (TMB peroxidase substrate, BioFx®,SurModics, Eden Prairie, Minn.) was added and incubated for 3 to 5minutes in the dark. The reaction was stopped by addition of 0.5M HCland the plate was read at 450 nm in a plate-reader.

Alternatively, To characterize recombinant expressed antibodies fortheir ability to preferentially bind ACTH 1-39 and not ACTH 1-13 or ACTH18-39 (respectively containing the amino acids contained in alpha-MSHand CLIP), a competition HTRF ELISA was performed. In parallel, 10 μl ofan antibody dilution series (highest final concentration of 100 nM) wereincubated with 10 μl of N-term biotinylated human ACTH 1-39 (67 nMfinal) alone or in combination with either (i) ACTH 1-13 (55 nM final)and ACTH 18-39 (55 nM final), or (ii) ACTH 1-13 (550 nM final) and ACTH18-39 (550 nM final) in a HTRF plate. Twenty microliters of Eu3+cryptate labeled anti-hu Fc donor and 20 μl of d2-labeled streptavidinacceptor were added to each well and incubated for 1 hour at roomtemperature. Fluorescence was measured at 620 and 665 nM with a delay of300 μsec.

Results

Using the above-described methods, numerous functional (antagonistic)antibodies that bind intact human ACTH, but which do not, or do notappreciably bind to alpha-MSH or CLIP were identified. Polypeptide andexemplary coding sequences of these antibodies are contained in theincluded biological sequence listing. Additionally, sequences for Ab13,Ab15, and Ab17 are shown in the included FIGS. 1-4. The full-lengthantibodies Ab13, Ab15, and Ab17 used in these examples were expressed asthe heavy chain polypeptides having the sequences of SEQ ID NOs: 1, 41,and 81, respectively, and the light chain polypeptides of SEQ ID NOs:21, 61, and 101, respectively. The heavy chain polypeptides ofantibodies Ab13, Ab15, and Ab17 were expressed from the polynucleotidesof SEQ ID NOs: 11, 51, and 91, respectively. The light chainpolypeptides of antibodies Ab13, Ab15, and Ab17 were expressed from thepolynucleotides of SEQ ID NOs: 31, 71, and 111, respectively.

The full-length antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab9, Ab10,Ab11, and Ab12 used in these examples were expressed as the heavy chainpolypeptides having the sequences of SEQ ID NOs: 681, 721, 761, 801,841, 881, 921, 961, 1001, 1041, and 1081, respectively, and the lightchain polypeptides of SEQ ID NOs: 701, 741, 781, 821, 861, 901, 941,981, 1021, 1061, and 1101, respectively. The heavy chain polypeptides ofantibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab9, Ab10, Ab11, and Ab12were expressed from the polynucleotides of SEQ ID NOs: 691, 731, 771,811, 851, 891, 931, 971, 1011, 1051, and 1091, respectively. The lightchain polypeptides of antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab9,Ab10, Ab11, and Ab12 were expressed from the polynucleotides of SEQ IDNOs: 711, 751, 791, 831, 871, 911, 951, 991, 1031, 1071, and 1111,respectively. Additional features of said antibodies are identified bySEQ ID NOs in FIGS. 5-12.

The binding and functional properties of exemplary anti-ACTH antibodiesproduced according to the invention are further described below.

FIG. 13 are representative of binding curves for the identifiedanti-ACTH antibodies for human ACTH (showing results for Ab1). EC50values were computed for each antibody based upon their binding curvesand are shown in Table 1 below. The results demonstrate that Ab1-Ab7,Ab9-Ab13, Ab15, and Ab17 bind to and recognize human ACTH with highaffinity, ranging between EC50 values of 0.24 nM and 2.24 nM.

TABLE 1 Binding (EC50) of anti-ACTH antibodies for human ACTH. ANTIBODYhuACTH 1-39 EC₅₀ nM Ab1 0.48 Ab2 0.42 Ab3 0.24 Ab4 0.39 Ab5 1.50 Ab62.00 Ab7 2.24 Ab9 2.05 Ab10 1.57 Ab11 0.81 Ab12 0.76 Ab13 1.04 Ab15 0.90Ab17 0.81

Additionally, anti-human ACTH antibodies that do not recognize ACTH 1-13(SEQ ID NO:1122) or ACTH 18-39 (SEQ ID NO:1124) were identified byELISA. Briefly, neutravidin plates (Thermo Scientific) were coated witha mixture of biotinylated ACTH 1-13 and ACTH 18-39 (50 μl per well, 1μg/ml each peptide) and the ELISA assay run as described above.

Results: FIG. 14 shows representative binding curves for an anti-ACTHantibody (specifically, Ab1) for ACTH 1-13 or ACTH 18-39. Based uponthese results, the EC50 was determined to be >10 μM in all instances, asshown in Table 2, indicating at most relatively low specific binding (orno specific binding).

TABLE 2 Binding (EC₅₀) of anti-ACTH antibodies for human ACTH 1-13 andhuman ACTH 18-39. huACTH huACTH ANTIBODY 1-13 ECso 18-39 EC₅₀ Ab1 >10μM >10 μM Ab2 >10 μM >10 μM Ab3 >10 μM >10 μM Ab4 >10 μM >10 μM Ab5 >10μM >10 μM Ab6 >10 μM >10 μM Ab7 >10 μM >10 μM Ab9 >10 μM >10 μM Ab10 >10μM >10 μM Ab11 >10 μM >10 μM Ab12 >10 μM >10 μM Ab13 >10 μM >10 μMAb15 >10 μM >10 μM Ab17 >10 μM >10 μM

Alternatively, to identify antibodies that preferentially bind ACTH 1-39(SEQ ID NO:1121) and not ACTH 1-13 (SEQ ID NO:1123) or ACTH 18-39 (SEQID NO:1124) sub-peptides of full length ACTH (i.e., corresponding to theamino acids contained in alpha-MSH and CLIP, respectively), acompetition HTRF ELISA was performed.

In parallel, 10 μl of an antibody dilution series (highest finalconcentration of 100 nM) were incubated with 10 μl of N-termbiotinylated human ACTH 1-39 (67 nM final) alone or in combination witheither (i) ACTH 1-13 (55 nM final) and ACTH 18-39 (55 nM final), or (ii)ACTH 1-13 (550 nM final) and ACTH 18-39 (550 nM final) in a HTRF plate.Twenty microliters of Eu3+ cryptate labeled anti-hu Fc donor and 20 μlof d2-labeled streptavidin acceptor were added to each well andincubated for 1 hour at room temperature. Fluorescence was measured at620 and 665 nM with a delay of 300 μsec.

Results

FIG. 15 and FIG. 45 provide representative binding data for the subjectanti-human ACTH antibodies to ACTH 1-39 and the inability of human ACTH1-13 and ACTH 18-39 to compete with binding of ACTH 1-39 (specifically,for Ab5 and Ab13, respectively). Similar lack of effects of human ACTH1-13 and ACTH 18-39 on binding to ACTH 1-39 were observed for Ab6-Ab7,Ab9-Ab13, Ab15, and Ab17 (not shown). The lack of effect of ACTH 1-13and ACTH 18-39 on binding to ACTH 1-39 is also reflected in the EC50values of >10 μM for these fragments indicated in Table 2 above. Theseresults demonstrate that Ab5-Ab7, Ab9-Ab13, Ab15, and Ab17 bind to ACTH1-39 but do not bind (or do not appreciably bind) ACTH 1-13 or ACTH18-39.

Humanized forms of antibodies Ab1, Ab2, Ab3, Ab4, Ab6, Ab7, Ab10, Ab11,Ab12, Ab13, Ab15, and Ab17 were produced and are identified by anappended “.H”, i.e., Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab6.H, Ab7.H, Ab10.H,Ab11.H, Ab12.H, Ab13.H, Ab15.H, and Ab17.H. Further variants of thehumanized Ab7.H and Ab11.H sequences were also produced and areidentified as Ab7A.H and Ab11A.H, respectively.

Functional Characterization of Antibodies by cAMP Assay

The ability of anti-ACTH antibodies to neutralize ACTH-induced MC2Rsignaling was tested in a cell-based assay.

For Ab1-Ab4, to identify antibodies that neutralize ACTH-inducedsignaling via MC2R, antibody solutions were incubated with ACTH 1-39 at3× the final concentration (100 pM) for 1 hour. While theantibody/antigen complexes were incubated, MC2R cells were detached with0.25% trypsin for 4 minutes. The cells were washed and re-suspended at2×10⁶ cells per ml in assay buffer (MSD) and treated with 0.2 mM IBMX.Ten microliters of cells was combined with 20 μl of antigen/antibodymixture and added to a cAMP plate (MSD) and incubated for 30 minutes atroom temperature while shaking. After the incubation, 20 μl of labeledcAMP in cell lysis buffer (MSD) was added and incubated 1 hour whileshaking. Following last incubation 100 μl of 1.5×MSD read buffer wasadded and read with Sector Imager 2400.

Results: FIG. 16 shows an inhibition curve (for Ab1) that isrepresentative of the inhibition curves obtained with the other testedantibodies. The inhibition results were quantified for each antibody toyield an IC50 value, which are summarized in Table 3 below. Theseresults demonstrated that anti-ACTH 1-39 antibodies Ab1-Ab4 inhibitedACTH induced cAMP in cells expressing MC2R.

TABLE 3 Inhibition (IC50) of ACTH induced cAMP in cells expressing MC2Rby anti-ACTH antibodies. ANTIBODY MC2R IC₅₀ nM Ab1 0.14 Ab2 0.25 Ab30.29 Ab4 0.46 Ab5 0.11 Ab6 0.03 Ab7 0.09 Ab9 0.12 Ab10 0.16 Ab11 0.03Ab12 0.05 Ab13 0.20 Ab15 0.14 Ab17 0.48 Ab1.H 0.01 Ab2.H 0.05 Ab3.H 0.15Ab4.H 0.03 Ab6.H 0.06 Ab7.H 0.11 Ab7A.H 0.09 Ab10.H 0.01 Ab11.H 0.02Ab11A.H 0.08 Ab12.H 0.05 Ab13.H 0.10 Ab15.H 0.60 Ab17.H 0.37

Alternatively, for Ab5-Ab7 and Ab9-Ab13, Ab15, Ab17, Ab1.H-Ab7.H,Ab7A.H, Ab10.H-Ab13.H, Ab11A.H, Ab15.H, and Ab17.H, to identifyantibodies that neutralize ACTH 1-39 induced signaling via MC2R,antibody solutions were incubated with ACTH (1-39) at 4× the finalconcentration (100 pM) for 1 hr. While the antibody/antigen complexeswere incubated for 1 hour, MC2R cells (Life Technologies) were detachedwith 0.25% trypsin for 4 minutes. The cells were washed and re-suspendedat 1×10⁶ cells per ml culture media. Twenty microliters of Ab/antigenmixture was mixed with 20 μl of cells in HTRF plates and incubated withshaking for 30 minutes. Twenty microliters of Eu3+ cryptate labeledanti-cAMP MAb and 20 μl d2-labeled cAMP was added to each well andincubated for 1 hour with shaking. Fluorescence was measured at 620 and665 nM with a delay of 300 μsec.

Results

FIG. 17 and FIG. 46 are representative of the inhibition curves obtainedby this method (results are shown for Ab5 and Ab13, respectively). Thecomputed IC50 values for each antibody (shown in Table 3 above)demonstrate that Ab5-Ab7, Ab9-Ab3, Ab15, Ab17, Ab1.H-Ab7.H, Ab7A.H,Ab10.H-Ab13.H, Ab11A.H, Ab15.H, and Ab17.H inhibited ACTH-induced cAMPin cells expressing MC2R.

Example 2

Binding Affinities of Anti-ACTH Antibodies

Binding affinities of monoclonal antibodies for human and mouse ACTHwere estimated using Surface Plasmon Resonance (SPR) on the ProteOn™XPR36 (Bio-Rad, Hercules, Calif.). Antibody was immobilized to thesurface of general amine coupling (GLC or GLM) Chips (Bio-Rad). Adilution series of human ACTH 1-39 (SEQ ID NO: 1121) prepared in1×HBS-EP+ Buffer (10 mM Hepes; 150 mM NaCl; 3 mM EDTA, 0.05% Polysorbate20; pH 7.6 at 25° C.) purchased from Thermo Scientific and supplementedwith 0.2 mg/mL Bovine Serum Albumin (BSA) from Jackson ImmunoResearchand 0.005% sodium azide from VWR was used to query the antibodies. Atthe chosen concentrations of antigen (ranging from 454 ng/ml to 5.6ng/ml) association times of 200 seconds and dissociation times of 30-200minutes were used with the ProteOn™ Manager Software (v3.1.0.6, Bio-Rad)to group and fit data using a 1:1 Langmuir binding model. Surfaces wereregenerated between analyte queries using 10 mM glycine at pH 2.0. Datarepeated across a single density was averaged and a single K_(D) andstandard propagation of error calculated for each antibody.

The same procedure was used to determine binding affinities ofantibodies for human alpha-MSH (ACTH 1-13) (SEQ ID NO:1123) and CLIP(ACTH 18-39) (SEQ ID NO:1124) except peptide concentrations ranged from1.66 μg/ml to 0.02 μg/ml and 2.46 μg/ml to 0.03 μg/ml respectively withan association time of 200 seconds and dissociation times of 1-10minutes.

The measured antibody affinities for ACTH are listed in Table 4.

TABLE 4 Antibody Ka (1/Ms) Kd (1/s) K_(D) (M) Ab1 1.0 × 10⁶ 1.9 × 10⁻⁴1.9 × 10⁻¹⁰ Ab2 1.0 × 10⁶ 1.3 × 10⁻⁴ 1.3 × 10⁻¹⁰ Ab3 8.2 × 10⁵ 1.5 ×10⁻⁵ 1.8 × 10⁻¹¹ Ab4 1.0 × 10⁶ 2.7 × 10⁻⁴ 2.7 × 10⁻¹⁰ Ab5 1.0 × 10⁶ 6.4× 10⁻⁵ 6.4 × 10⁻¹¹ Ab6 1.0 × 10⁶ 1.9 × 10⁻⁵ 1.9 × 10⁻¹¹ Ab7 1.0 × 10⁶3.7 × 10⁻⁵ 3.7 × 10⁻¹¹ Ab9 9.1 × 10⁵ 4.7 × 10⁻⁵ 5.2 × 10⁻¹¹ Ab10 1.0 ×10⁶ 1.1 × 10⁻⁴ 1.1 × 10⁻¹⁰ Ab11 1.0 × 10⁶ 4.0 × 10⁻⁵ 4.0 × 10⁻¹¹ Ab128.2 × 10⁵ 9.8 × 10⁻⁵ 1.2 × 10⁻¹⁰ Ab13 1.0 × 10⁶ 8.3 × 10⁻⁵ 8.3 × 10⁻¹¹Ab15 8.3 × 10⁵ 7.2 × 10⁻⁵ 8.8 × 10⁻¹¹ Ab17 7.6 × 10⁵ 2.5 × 10⁻⁴ 3.3 ×10⁻¹⁰ Ab1.H 8.0 × 10⁵ 5.1 × 10⁻⁵ 6.3 × 10⁻¹¹ Ab2.H 8.9 × 10⁵ 1.6 × 10⁻⁴1.8 × 10⁻¹⁰ Ab3.H 9.4 × 10⁵ 1.6 × 10⁻⁵ 1.7 × 10⁻¹¹ Ab4.H 1.0 × 10⁶ 1.3 ×10⁻⁴ 1.3 × 10⁻¹⁰ Ab6.H 8.9 × 10⁵ 2.6 × 10⁻⁵ 2.9 × 10⁻¹¹ Ab7.H 1.0 × 10⁶5.2 × 10⁻⁵ 5.2 × 10⁻¹¹ Ab7A.H 1.0 × 10⁶ 6.0 × 10⁻⁵ 6.0 × 10⁻¹¹ Ab10.H1.0 × 10⁶ 1.7 × 10⁻⁵ 1.7 × 10⁻¹¹ Ab11.H 6.4 × 10⁵ 1.4 × 10⁻⁵ 2.2 × 10⁻¹¹Ab11A.H 7.4 × 10⁵ 6.0 × 10⁻⁵ 8.2 × 10⁻¹¹ Ab12.H 3.7 × 10⁵ 5.6 × 10⁻⁵ 1.5× 10⁻¹⁰ Ab13.H 9.0 × 10⁵ 6.8 × 10⁻⁵ 7.6 × 10⁻¹¹ Ab15.H 1.0 × 10⁶ 1.6 ×10⁻⁴ 1.6 × 10⁻¹⁰ Ab17.H 8.2 × 10⁵ 1.4 × 10⁻⁵ 1.7 × 10⁻¹⁰

Examples of antibody affinities for CLIP are listed in Table 5.

TABLE 5 Antibody Ka (1/Ms) Kd (1/s) K_(D) (M) Ab1 6.2 × 10⁵ 9.2 × 10⁻²1.5 × 10⁻⁷ Ab2 8.4 × 10⁵ 2.6 × 10⁻² 3.1 × 10⁻⁸ Ab3 3.4 × 10⁵ 8.5 × 10⁻³2.5 × 10⁻⁸ Ab4 7.1 × 10⁵ 1.9 × 10⁻¹ 2.7 × 10⁻⁷ Ab5 <1.0 × 10⁰   >1.0 ×10⁻¹   >1.0 × 10⁻¹   Ab6 <1.0 × 10⁰   >1.0 × 10⁻¹   >1.0 × 10⁻¹   Ab7<1.0 × 10⁰   >1.0 × 10⁻¹   >1.0 × 10⁻¹   Ab9 <1.0 × 10⁰   >1.0 ×10⁻¹   >1.0 × 10⁻¹   Ab10 1.1 × 10⁶ 2.7 × 10⁻¹ 2.5 × 10⁻⁷ Ab11 1.6 × 10⁶8.6 × 10⁻² 5.4 × 10⁻⁸ Ab12 8.9 × 10⁵ 2.4 × 10⁻² 2.7 × 10⁻⁸ Ab13 9.8 ×10⁵ 8.5 × 10⁻³ 8.6 × 10⁻⁹ Ab15 6.3 × 10⁵ 3.1 × 10⁻² 5.0 × 10−8 Ab17 5.1× 10⁵ 3.1 × 10⁻³ 6.1 × 10⁻⁹ Ab1.H 5.8 × 10⁵ 1.2 × 10⁻² 2.0 × 10⁻⁸ Ab2.H6.0 × 10⁵ 1.7 × 10⁻² 2.8 × 10⁻⁸ Ab3.H 3.2 × 10⁵ 5.3 × 10⁻³ 1.6 × 10⁻⁸Ab4.H 2.5 × 10⁵ 2.3 × 10⁻² 9.2 × 10⁻⁸ Ab6.H <1.0 × 10⁰   >1.0 ×10⁻¹   >1.0 × 10⁻¹   Ab7.H <1.0 × 10⁰   >1.0 × 10⁻¹   >1.0 × 10⁻¹  Ab7A.H <1.0 × 10⁰   >1.0 × 10⁻¹   >1.0 × 10⁻¹   Ab10.H 5.4 × 10⁵ 7.0 ×10⁻³ 1.3 × 10⁻⁸ Ab11.H 5.4 × 10⁵ 1.1 × 10⁻² 2.0 × 10⁻⁸ Ab11A.H 7.0 × 10⁵1.4 × 10⁻² 2.0 × 10⁻⁸ Ab12.H 5.8 × 10⁵ 5.1 × 10⁻³ 8.8 × 10⁻⁹ Ab13.H 5.0× 10⁵ 7.0 × 10⁻³ 1.4 × 10⁻⁸ Ab15.H 4.2 × 10⁵ 9.3 × 10⁻² 2.2 × 10⁻⁷Ab17.H 6.6 × 10⁵ 8.7 × 10⁻³ 1.3 × 10⁻⁸

Examples of antibody affinities for alpha-MSH are listed in Table 6.

TABLE 6 Antibody Ka (1/Ms) Kd (1/s) K_(D) (M) Ab1 <1.0 × 10⁰   <1.0 ×10⁻¹   <1.0 × 10⁻¹   Ab2 <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab3<1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab4 <1.0 × 10⁰   <1.0 × 10⁻¹  <1.0 × 10⁻¹   Ab5 2.6 × 10⁵ 1.4 × 10⁻² 5.5 × 10⁻⁸ Ab6 3.3 × 10⁵ 5.2 ×10⁻³ 1.6 × 10⁻⁸ Ab7 1.3 × 10⁵ 1.3 × 10⁻² 5.4 × 10⁻⁸ Ab9 9.0 × 10⁵ 9.0 ×10⁻³ 6.3 × 10⁻⁸ Ab10 <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab11 <1.0× 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab12 <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0× 10⁻¹   Ab13 <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab15 <1.0 × 10⁰  <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab17 <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 ×10⁻¹   Ab1.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab2.H <1.0 × 10⁰  <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab3.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 ×10⁻¹   Ab4.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab6.H 2.4 × 10⁵4.0 × 10⁻³ 1.6 × 10⁻⁸ Ab7.H 2.5 × 10⁵ 9.4 × 10⁻³ 3.7 × 10⁻⁸ Ab7A.H 2.7 ×10⁵ 1.3 × 10⁻² 4.8 × 10⁻⁸ Ab10.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 ×10⁻¹   Ab11.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab11A.H <1.0 ×10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab12.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0× 10⁻¹   Ab13.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab15.H <1.0 ×10⁰   <1.0 × 10⁻¹   <1.0 × 10⁻¹   Ab17.H <1.0 × 10⁰   <1.0 × 10⁻¹   <1.0× 10⁻¹  

Example 3 Inhibition of ACTH-Induced Signaling Via MC1R

CHO-K1 cells expressing MC1R with a beta-lactamase reporter gene underthe control of a cAMP response element (Life Technologies) were used ina GeneBLAzer FRET cell based assay. Cells were grown in DMEMsupplemented with 10% dialyzed FBS, 10 mM glutamax, 0.1 mM non-essentialamino acids, 25 mM HEPES, and 600 ug/ml Hygromycin. The day before theassay the cells were detached with 0.25% trypsin, counted using ahemacytometer and adjusted to 2×10⁵ cells/ml in growth media. 100ul/well was plated in a 96-well black wall clear bottom plate. On theday of the assay anti-ACTH antibody dilutions starting at 40 nM wereincubated in the presence of 5 nM ACTH (American Peptide) for 1 hr at 37C. The media was removed from the MC1R cells and replaced with assaymedia alone, supplemented with ACTH, or ACTH incubated in the presenceof the various antibody dilutions. All conditions were performed induplicate. The cells were incubated for 4 hours and then loaded with 20μl 6× substrate loading solution (Life Technologies) for 2 hours andread at an excitation wavelength of 409 nm and emission wavelengths 460and 530 nm. The ratio of blue (460 nm) to green (530 nm) was used forplotting.

Results

FIG. 18 and FIG. 47 are representative of the inhibition curves obtainedby this method (results are shown for Ab1 and Ab13, respectively). Thecomputed IC50 values for each antibody (shown in Table 7, below)demonstrate that Ab1-Ab7 and Ab9-Ab13, Ab15, Ab17, Ab1.H-Ab7.H, Ab7A.H,Ab10.H-Ab13.H, Ab11A.H, Ab15.H, and Ab17.H inhibited ACTH-induced cAMPin cells expressing MC1R.

TABLE 7 Inhibition (IC50) of ACTH induced cAMP in cells expressing MC1Rby anti-ACTH antibodies. ANTIBODY MC1R IC₅₀ nM Ab1 2.38 Ab2 3.62 Ab34.12 Ab4 5.73 Ab5 1.96 Ab6 1.04 Ab7 1.29 Ab9 1.32 Ab10 2.14 Ab11 1.49Ab12 1.66 Ab13 2.06 Ab15 2.11 Ab17 2.56 Ab1.H 1.36 Ab2.H 2.67 Ab3.H 2.06Ab4.H 2.27 Ab6.H 1.83 Ab7.H 1.64 Ab7A.H 1.19 Ab10.H 0.54 Ab11.H 1.37Ab11A.H 0.95 Ab12.H 1.99 Ab13.H 1.32 Ab15.H 2.10 Ab17.H 1.56

Example 4 Inhibition of ACTH-Induced Signaling Via MC3R, MC4R and MC5R

Methods

For Ab1-Ab7 and Ab9, CHO-K1 cells expressing MC3R, MC4R or MC5R with areporter gene under the control of a cAMP response element (LifeTechnologies) were used in a Meso Scale Discovery assay measuring cAMP.Cells were grown in DMEM supplemented with 10% dialyzed FBS, 10 mMglutamax, 0.1 mM non-essential amino acids, 25 mM HEPES, 5 μg/mlblasticidin and 600 μg/ml Hygromycin (MC3R), 100 g/ml Zeocin (MC4R) or400 μg/ml Hygromycin (MC5R). The day of the assay the cells weredetached with 5 mM EDTA, counted using a hemacytometer and adjusted to2×10⁶ cells/ml in Hepes buffered saline plus MgCl2, pH 7.3 (assaybuffer). A 1:2 dilution series of anti-ACTH antibodies were incubated inthe presence of ACTH (American Peptide or Bachem) for 1 hour at 37° C.For MC3R and MC4R, antibody concentrations started at 833 nM and ACTHwas used at 100 nM. For MC5R, antibody concentrations started at 17 μMand ACTH was used at 5 μM. Twenty microliters of the assay buffer, ACTHor antibody/ACTH mixture was then added to the assay plate, followed by10 μl of cells. After a 30 minute incubation at room temperature withshaking, the cells were lysed with 20 μl assay buffer plus Triton X-100supplemented with 2.5 nM TAG-cAMP for 1 hour at room temperature withshaking. Finally 100 μl of 1.5× Read buffer T was added to each well andread on a Sector Imager 2400.

For Ab10-Ab3, Ab15, Ab17, Ab1.H-Ab7.H, Ab7A.H, Ab10.H-Ab13.H, Ab11A.H,Ab15.H, and Ab17.H, to identify antibodies that neutralize ACTH 1-39induced signaling via MC3R or MC4R, antibody solutions were incubatedwith ACTH (1-39) at 4× the final concentration (250 nM) for 1 hr. Whilethe antibody/antigen complexes were incubated for 1 hour, MC3R or MC4Rcells (Life Technologies) were detached with 0.25% trypsin for 4minutes. The cells were washed and re-suspended at 1×10⁶ cells per mlculture media. Twenty microliters of Ab/antigen mixture was mixed with20 μl of cells in HTRF plates and incubated with shaking for 30 minutes.Twenty microliters of Eu3+ cryptate labeled anti-cAMP MAb and 20 μld2-labeled cAMP was added to each well and incubated for 1 hour withshaking. Fluorescence was measured at 620 and 665 nM with a delay of 300μsec.

Also Ab10-Ab13, Ab15, Ab17, Ab1.H-Ab7.H, Ab7A.H, Ab10.H-Ab13.H, Ab11A.H,Ab15.H, and Ab17.H, to identify antibodies that neutralize ACTH 1-39induced signaling via MC5R, antibody solutions were incubated with ACTH(1-39) at 4× the final concentration (10 uM) for 1 hr. While theantibody/antigen complexes were incubated for 1 hour, MC5R cells (LifeTechnologies) were detached with 0.25% trypsin for 4 minutes. The cellswere washed and re-suspended at 1×10⁶ cells per ml culture media. Twentymicroliters of Ab/antigen mixture was mixed with 20 μl of cells in HTRFplates and incubated with shaking for 30 minutes. Twenty microliters ofEu3+ cryptate labeled anti-cAMP MAb and 20 μl d2-labeled cAMP was addedto each well and incubated for 1 hour with shaking. Fluorescence wasmeasured at 620 and 665 nM with a delay of 300 μsec.

Results

FIGS. 19, 20, and 21 are representative of the observed antibodyinhibition of ACTH induced cAMP in cells expressing MC3R, MC4R, and MC5Rrespectively (results are shown for Ab1). FIGS. 48, 49, and 50 arerepresentative of the observed antibody inhibition of ACTH induced cAMPin cells expressing MC3R, MC4R, and MC5R respectively (results are shownfor Ab13). The computed IC50 values for each antibody (shown in Table 8,below) demonstrate that Ab1-Ab7 and Ab9-Ab13, Ab15, Ab17, Ab1.H-Ab7.H,Ab7A.H, Ab10.H-Ab13.H, Ab11A.H, Ab15.H, and Ab17.H inhibitedACTH-induced cAMP in cells expressing MC3R, MC4R, and MC5R.

TABLE 8 Inhibition (IC50) of ACTH induced cAMP in cells expressing MC3R,MC4R, and MC5R by anti-ACTH antibodies. MC3R MC4R MC5R ANTIBODY IC₅₀ nMIC₅₀ nM IC₅₀ μM Ab1 101.0  56.4 1.1 Ab2 79.0 54.5 1.1 Ab3 58.7 54.2 1.1Ab4 113.0  65.8 1.3 Ab5 58.1 43.4 1.0 Ab6 62.8 55.2 1.0 Ab7 64.2 49.71.1 Ab9 55.7 50.6 1.1 Ab10 133.2  66.3 5.4 Ab11 108.3  49.4 4.2 Ab1299.7 50.6 5.4 Ab13 141.3  71.4 4.7 Ab15 135.9  93.9 6.1 Ab17 138.9  94.45.9 Ab1.H 83.9 43.7 3.6 Ab2.H 65.6 46.3 2.4 Ab3.H 70.6 34.8 3.5 Ab4.H87.7 41.8 3.1 Ab6.H 89.6 52.2 3.9 Ab7.H 94.4 49.3 4.7 Ab7A.H 92.3 55.9not determined Ab10.H 104.3  50.6 3.5 Ab11.H 57.8 33.8 3.8 Ab11A.H 59.135.9 3.1 Ab12.H 78.2 46.9 3.7 Ab13.H 64.9 46.9 3.1 Ab15.H 131.3  79.54.3 Ab17.H 109.9  81.6 4.0

Example 5 Inhibition of ACTH-Induced Cortisol Secretion by Y1 Cells

The Y-1 cell line (mouse adrenal cell line) (ATCC) secretes cortisol inresponse to ACTH stimulation. Cells were grown on collagen coated flasksin Ham's F-12K media supplemented with 15% Horse Serum and 2.5% FBS.Cells at 400,000 cells/ml were seeded at 100 μl per well into a collagencoated clear bottom black walled 96 well plate (Costar) and incubatedovernight. The media was then changed to F12K supplemented with 1% BSA(assay media) and cells incubated overnight. Assay media supplementedwith 3 nM ACTH (American Peptide or Bachem) was incubated in thepresence of anti-ACTH antibody (1:3 dilution series starting at 81 nM)at 37° C. for 1 hour. The media was removed from the Y-1 cells andreplaced with assay media alone, supplemented with ACTH, or ACTHincubated in the presence of the various antibodies. Treatment of thecells was for 24 hrs. The experimental media was removed from cells,diluted 1:10 and the cortisol level was determined with Cortisolparameter assay kit (R&D, Minneapolis, Minn.). Briefly microplate stripswere incubated with 50 μl Primary Antibody solution (except non-standardbinding wells) for 1 hour at room temperature with shaking. Plate wasthen washed 4× with 400 μl/well wash buffer. Then 100 μl standards andsamples were added to the plate, followed by 50 μl cortisol conjugate.Plates were incubated 2 hours at room temperature with shaking and thenwashed as above. The plates were developed with 200 μl/well substratesolution for 30 minutes, followed by the addition of 50 μl/well stopsolution. Plates were read at 450 nm with a 570 nm correction.

Results

FIG. 22 and FIG. 51 are representative of the observed antibodyinhibition of ACTH induced cAMP in Y1 cells (results are shown for Ab1and Ab13, respectively). The computed IC50 values for each antibody(shown in Table 9, below) demonstrate that Ab1-Ab7 and Ab9-Ab13, Ab15,Ab17, Ab1.H-Ab7.H, Ab7A.H, Ab10.H-Ab13.H, Ab11A.H, Ab15.H, and Ab17.Hinhibited ACTH-induced cortisol in Y1 cells.

TABLE 9 Inhibition (IC50) of ACTH induced cortisol in Y1 cells byantibodies Ab1-Ab7 and Ab9-Ab12. ANTIBODY Y1 Cells IC₅₀ nM Ab1 2.36 Ab22.35 Ab3 7.72 Ab4 17.19 Ab5 3.49 Ab6 1.44 Ab7 2.49 Ab9 3.47 Ab10 5.98Ab11 1.53 Ab12 2.68 Ab13 2.35 Ab15 1.84 Ab17 4.47 Ab1.H 1.77 Ab2.H 1.96Ab3.H 4.04 Ab4.H 2.43 Ab6.H 1.62 Ab7.H 2.05 Ab7A.H 2.26 Ab10.H 1.06Ab11.H 0.97 Ab11A.H 2.53 Ab12.H 4.13 Ab13.H 1.91 Ab15.H 3.83 Ab17.H 4.30

Example 6 Reduction of Corticosterone Levels in Mice by Anti-ACTHAntibodies

A pharmacodynamics study was conducted in female C57BL/6 mice. Five micewere injected with buffer and groups of 10 mice were dosed with either10 mg/kg of a control antibody of the same isotype (AD26-10), Ab2 orAb3. Injections were performed by IV (tail vein) bolus administration ondays 1 and day 7.

Blood samples were collected 24 hours before injection of test article(day 0), day 3, day 9 and day 12 in K₃EDTA tubes and processed to plasmafor corticosterone analysis. All samples were stored at −70° C.

Corticosterone levels in mouse plasma samples were assessed using aCorticosterone EIA kit (Enzo Life Sciences) according to manufacturer'sprotocol. Briefly 100 μl plasma samples are diluted 1:20, standards andcontrols were added to assay plate, followed by 50 μl of an alkalinephosphatase conjugated corticosterone and 50 μl of a polyclonal Ab tocorticosterone. Assay plate was incubated 2 hours at room temperaturewith shaking and then washed. It was developed with p-Npp for 1 hour andthen read at 405 with a 570 nm subtraction.

Results: FIGS. 23-26 demonstrate that Ab2 and Ab3 decrease plasmacorticosterone levels in mice.

Example 7 Reduction of Corticosterone Levels in Rats by Anti-ACTHAntibodies

A pharmacodynamics study was conducted in male Lewis rats. On day 1,rats were implanted with an Alzet pump (Durect #2ML1, 10 ul/hr for 7days) delivering either vehicle or rat ACTH (Bachem) at a rate of 0.05mg/kg/day. Twenty-four hours after pump implantation, the rats wereinjected with either 10 mg/kg of a control isotype antibody (AD26-10) orAb6. Injections were performed by IV (tail vein) bolus administration.The study was terminated 6 days post antibody injection.

Body weights were recorded daily and blood samples were collected on day0, 2, 3, 5, 7, and 8 in K₃EDTA tubes and processed to plasma forcorticosterone and aldosterone analysis. All samples were stored at −70°C.

Corticosterone levels in rat plasma samples were assessed using aCorticosterone EIA kit (Enzo Life Sciences) according to manufacturer'sprotocol. Briefly 100 μl plasma samples were diluted 1:20, standards andcontrols were added to assay plate, followed by 50 μl of an alkalinephosphatase conjugated corticosterone and 50 μl of a polyclonal Ab tocorticosterone. The assay plate was incubated 2 hours at roomtemperature with shaking and then washed. It was developed with p-Nppfor 1 hour, then stopped and read at 405 with a 570 nm subtraction.

Results

FIG. 27 demonstrates Ab6 inhibited ACTH induced weight loss. A one-wayanalysis of variance (ANOVA) was performed. Plasma corticosterone andaldosterone levels at day 0 (before antibody administration or pumpimplantation) are shown in FIG. 28 and FIG. 34, respectively. Plasmacorticosterone and aldosterone levels at day 2 (24 hours post pumpimplantation but pre-Ab dosing) are shown in FIG. 29 and FIG. 35,respectively. The results show that Ab6 reduced corticosterone (FIGS.30-33) and aldosterone (FIGS. 36-39) levels at days 3, 5, 7, and 8, withstatistically significant reductions observed in both corticosterone andaldosterone at days 3, 5, and 7, and at day 8 for aldosterone. AMann-Whitney two-tailed P value analysis was performed comparing groupsto the ACTH/AD26-10 group. Statistical significance values are as shownin the figures.

It was observed in some experiments that corticosterone levels variedfrom day to day, which was thought to result from varying levels ofstress, e.g., as a result of handling the animals. Notwithstanding,consistent differences were observed between the control and treatmentgroups (as well as statistically significant differences between them),indicating effectiveness of the antibody at neutralizing ACTH activityin vivo.

Example 8

Epitope Mapping of Anti-ACTH Antibodies

ACTH peptides were synthesized with a single point mutation in eachposition replacing the native amino acid with an Alanine (Ala). Inpositions 27, 32 and 34 the native Ala was replaced with Valine (Val).Per the usual convention these mutants are identified by the position inACTH 1-39 followed by the letter code for the substituted amino acid,e.g., 7A indicates ACTH 1-39 substituted with alanine at amino acidposition 7. Binding of monoclonal antibodies for human ACTH and eachmutant peptide was detected using Surface Plasmon Resonance (SPR) on theProteOn™ XRP36 (Bio-Rad, Hercules, Calif.). Samples and sample controlswere immobilized onto a GLC sensor chip at a single density usingstandard amine coupling. The running buffer for immobilization consistedof 1×HBS-EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Polysorbate 20,pH 7.6) and was carried out at 25 degrees C. The GLC chip wasinitialized and pre-conditioned per the manufacturer's protocol(bi-directional injections of 0.5% SDS, 50 mM NaOH, 100 mM HCl). Theimmobilization process was carried out step-wise to ensure a uniqueantibody on the spots of the ProteOn™ Chip. Activation of the surfacewas by a 1:1 mixture of EDAC/NHS and flow rate of 30 uL/min×6 minutes.Antibody samples were previously dialyzed or exchanged to 10 mM HEPES150 mM NaCl pH 7.2 and the antibody concentration was quantified using aNanodrop™2000 spectrophotometer (ThermoScientific). The immobilizationtargeted 2000-3000 RU. Antibody samples (10 ug/mL) in 10 mM SodiumAcetate, pH 5.5 were flowed at 30 uL/min×6 minutes. Deactivation wasdone at a flow rate of 30 uL/min for 6 minutes using 0.5M Ethanolamineconcomitantly with the next activation.

Following immobilization, the running buffer was changed to 1×HBS-EP+with BSA (0.2 mg/mL) (as a carrier) and Sodium Azide (4 uM) (as apreservative) and the chip surface was allowed to re-equilibrate with aninjection of new running buffer. Stock solutions of human ACTH peptide(1-39) and alanine/valine mutant peptides (Molecular Weight(s): 4.5 kD)at (1 mg/mL) were added to the running buffer at concentrations of 0.45μg/mL (100 nM) and used to query individual spots on the chip surfacewith flow rates of 100 uL/min×2 minutes and allowed to dissociate for1000 seconds. Regeneration of surfaces between analytes was accomplishedwith Glycine 10 mM at pH 2.0. The tested antibodies were either theoriginal rabbit sequence or humanized sequence variants of each of thesubject antibodies, specifically, Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab5,Ab6.H, Ab7.H, Ab9, Ab10.H, Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15, andAb17. The “.H” appended to a given antibody name indicates a humanizedvariant of the identified antibody. Ab11A.H is variant of Ab11.Hcontaining a sequence difference within one of the CDRs, which wasobserved to cause a slight difference in epitope binding (one amino aciddifference). Because the humanization process generally retains thebinding specify of the antibody to the target the tested antibodies areinterpreted to bind to the same epitopes as their respective parentantibodies.

Sensorgrams representing affinity data of mutant peptide binding to apanel of antibodies were assessed via multiple measures. A visualinspection was first performed for each sensorgram to assess apparentmaximal response (Rmax) relative to the native ACTH peptide (1-39).Second, a visual inspection of the dissociation phase was performed withan emphasis on the curve shape relative to the native ACTH peptide.Off-rates were calculated for native ACTH peptide and binding of eachmutant peptide to the panel of antibodies. Finally, to confirm theintegrity of each peptide reagent, each member of the peptide librarywas individually assessed to a broad panel of antibodies to ensure eachpeptide displayed binding activity similar to the native peptide to atleast one antibody. The determination of amino acids residues importantfor antibody binding were made based on the collective assessment of allparameters described.

Results

Binding and dissociation curves are shown in FIG. 40A-O for binding ofantibodies Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab5, Ab6.H, Ab7.H, Ab9, Ab10.H,Ab11.H, Ab11A.H, Ab12.H, Ab13.H, Ab15, and Ab17. The upper panel showsthe binding curves for positions important for antibody binding (labeledat the right end, e.g., “21A” indicates the binding curve for thealanine scan mutant containing Alanine at position 21). The lower panelshows the binding of the remaining mutant positions, i.e., thosedetermined not to be important for antibody binding. For reference, bothpanels also show the binding curve for wild-type ACTH (labeledhuACTH(1-39)).

FIG. 41 tabulates the effects of all of the ACTH mutants on antibodybinding. The positions listed in each column identify the alaninescanning mutants that were determined to be important for antibodybinding; these are shown in order of position in order to illustrate thespatial arrangement of the residues along the ACTH primary sequence. Thepositions important for antibody binding were interpreted to jointlymake the epitopes bound by each antibody. Based on these results, theepitopes bound by each antibody were concluded to be as follows:

Ab1 or Ab1.H: epitope containing residues 16, 18, and 20-23 of humanACTH.

Ab2 or Ab2.H: epitope containing residues 16, 18, and 20-23 of humanACTH.

Ab3 or Ab3.H: epitope containing residues 16, 18, and 20-23 of humanACTH.

Ab4 or Ab4.H: epitope containing residues 16, 18, and 20-23 of humanACTH.

Ab5: epitope containing residues 7-11, 13-14, and 18-19 of human ACTH.

Ab6 or Ab6.H: epitope containing residues 7-11, 13-14, 16, 18-19, and 23of human ACTH.

Ab7 or Ab7.H: epitope containing residues 7-11, 13-14, and 18-19 ofhuman ACTH.

Ab9: epitope containing residues 7-11, 14, and 18 of human ACTH.

Ab10 or Ab10.H: epitope containing residues 16, 18, and 20-23 of humanACTH.

Ab11 or Ab11.H: epitope containing residues 16-18 and 20-23.

Ab11A.H: epitope containing residues 16-23 of human ACTH.

Ab12 or Ab12.H: epitope containing residues 16-23 of human ACTH.

Ab13 or Ab13.H: epitope containing residues 17-23 of human ACTH.

Ab15: epitope containing residues 17-23 of human ACTH.

Ab17: epitope containing residues 17-23 of human ACTH.

From these results it was further noted that the antibodies can bedivided into two groups based upon the amount of overlap between theresidues forming the epitope. One group contains antibodies Ab1-Ab4,Ab10-Ab13, Ab15, Ab17, Ab1.H-Ab4.H, Ab10.H, Ab11.H, Ab11A.H, and Ab13.Hthat each bind to residues 16, 18, and 20-23 of human ACTH, andoptionally further bind to residues 17 and/or 19. The second groupincludes antibodies Ab5-Ab7 and Ab9 that each bind to residues 7-11, 14,and 18 of human ACTH, and optionally further bind to residues 13, 16,and/or 19. From these results it was concluded that an antibody thatbinds to the same epitope as any of these antibodies, or overlaps inbinding with residues of either or both of these epitopes, would likelyhave similar biological activity as the subject antibodies, includingthe ability to block MCR activation and inhibit the release of cortisoland aldosterone in vivo. Additionally, antibodies that bind to theseepitopes or a subset of residues thereof are predicted to resemble thesubject antibodies in their binding affinity characteristics (e.g.,selectivity or differential binding ability), including exhibitingstronger affinity for ACTH than for alpha-MSH or CLIP (such as at least10-fold, at least 100-fold, or at least 1000-fold stronger affinity forhuman ACTH than for alpha-MSH or CLIP or for both alpha-MSH and CLIP,i.e., a numerically lower K_(D) for ACTH than for alpha-MSH or CLIP byat least 10-fold, at least 100-fold, or at least 1000-fold).

Example 9 Anti-ACTH Antibodies Inhibit Binding of ACTH to MC2R

Inhibition of ACTH binding to the melanocortin-2 receptor (MC2R) wasdetermined using ACTH (1-39) 23 TYR, [125I] (Perkin Elmer) and an MC2Rtransfected cell line (Invitrogen). Briefly, MC2R cells were cultured tologarithmic growth in DMEM containing 10% dialyzed FBS, L-glutamine,NEAA, and HEPES. Selection pressure for MC2R expression was maintainedon the cells using Blasticidin, Zeocin, and Hygromycin at 5, 100, and600 μg/ml, respectively. Cells were harvested and plated on Perkin ElmerCytostar-T™ Scintillating Microplates at 4×10⁴ cells/well in 100 μL ofmedia and incubated at 35-38° C. in 5% CO₂ for 18-24 hours. Followingincubation cells were aspirated of media and 100 μL of DMEM containing2% BSA (DMEM-BSA) was added to each well. Cells were incubated until thetreatment solution was prepared.

The ¹²⁵I-ACTH tracer solution was prepared by adding 40 μL of the ACTH(1-39) 23 TYR, [125I] to 10 ml of DMEM-BSA (final concentration withcell 6.4 pM). Each antibody to be evaluated was prepared as a 1 mg/mlintermediate stock in DMEM-BSA from a 5 mg/ml master stock. Eachantibody solution (20 μl) and ¹²⁵I-ACTH tracer (480 μl) were combinedand incubated for 30 minutes at 35-38° C. Cells were aspirated andincubated in the presence of ¹²⁵I-ACTH tracer (Max binding), ¹²⁵I-ACTHtracer+ antibody, or ¹²⁵I-ACTH tracer+ACTH, 1 μM (ACTH control) for 1hour at 35-38° C. in 5% CO₂. Nonspecific background binding wasdetermined by adding the ¹²⁵I-ACTH tracer to cell-free wells(Background). At the end of incubation period wells were analyzed for¹²⁵I-ACTH tracer binding using a MicroBeta® Trilux (Perkin Elmer) todetermine the calculated counts per minute of each well.

Results

FIG. 42 shows that all anti-ACTH antibodies completely inhibited ACTHbinding to MC2R (similar to the background level measured in the absenceof cells, which is shown in the second bar from the left) within thelimits of detection of the assay. As expected, three negative controlantibodies (three rightmost bars) fail to inhibit ACTH binding asindicated by similar to levels detected in the absence of antibody(leftmost bar). The third to fourteenth columns from left to right inthe bar graph correspond to the results for the tested antibodies.

These results indicate that the mechanism by which the subject anti-ACTHantibodies inhibit activation of MC2R is by preventing binding of ACTHto this receptor. From these results it is predicted that activation ofthe other MCRs (MC1R, MC3R, MC4R, and MC5R) is by a similar mechanism,i.e., by decreasing or abolishing ACTH binding to the MCRs.

Example 10. Recognition of ACTH 1-24 by Recombinant Antibodies by ELISA

ACTH is a 39 amino acid peptide but analyses of various truncated ACTHpeptides have demonstrated ACTH 1-24 has full agonist activity of MC2R(Chen et al., Biochemistry 2007; 46 (40): 11389-11397). The peptidesequence of ACTH 1-24 is fully conserved (100% identity) among mammalianspecies including human (SEQ ID NO: 1122), horse (Equus przewalskii,NCBI Accession No. XP_008513480), cat (Felis catus, NCBI Accession No.XP_003984482), and dog (Canus lupus familiaris, NCBI accession no.AAK08973). In Example 9, above, it was demonstrated that each of thetested antibodies recognized ACTH epitopes exclusively contained in ACTH1-24. Additionally, Ab1-Ab7 and Ab9 bind ACTH 1-24 with similar affinityto ACTH 1-39 (data not shown). Taken together, these results stronglysuggest that the subject anti-ACTH antibodies would be able to bind tothe conserved ACTH 1-24 sequence within of horse, dog, and cat ACTH andthereby inhibit biological activities of ACTH in these species. This wasfurther assessed by determining whether the anti-ACTH antibodies couldblock MC2R receptor activation by the ACTH 1-24 peptide sequence that is100% conserved among humans, horses, dogs, and cats.

Methods

To assess neutralization of ACTH 1-24 induced signaling via MC2R,antibody solutions were incubated with ACTH (1-24) at 4× the finalconcentration (600 pM) for 1 hr. While the antibody/antigen complexeswere incubated for 1 hour, MC2R cells (Life Technologies) were detachedwith 0.25% trypsin for 4 minutes. The cells were washed and re-suspendedat 1×106 cells per ml culture media. Twenty microliters of Ab/antigenmixture was mixed with 20 μl of cells in HTRF plates and incubated withshaking for 30 minutes. Twenty microliters of Eu3+ cryptate labeledanti-cAMP MAb and 20 μl d2-labeled cAMP was added to each well andincubated for 1 hour with shaking. Fluorescence was measured at 620 and665 nM with a delay of 300 μsec.

Results

FIG. 43 and FIG. 44 shows an inhibition curves (for Ab2 and Ab13.H,respectively) that are representative of the inhibition curves obtainedwith the other tested antibodies. The inhibition results were quantifiedfor each antibody to yield an IC50 value, which are summarized in Table10 below. These results demonstrated that anti-ACTH antibodies Ab2,Ab2.H, Ab3, Ab3.H, Ab6, Ab6.H, Ab13, and Ab13.H inhibited ACTH 1-24induced cAMP in cells expressing MC2R. Notably, the antibodies testedwere representative of the two different epitope groups identified inExample 9, indicating that the antibodies of either group would havesimilar therapeutic activity in veterinary applications.

TABLE 10 IC50 (nM) for antibody inhibition of MC2R receptor activationby the ACTH 1-24 peptide. Antibody IC50 (nM) Ab2 1.3 Ab2.H 0.6 Ab3 0.8Ab3.H 0.4 Ab6 0.1 Ab6.H 0.1 Ab13 1.0 Ab13.H 0.3

Example 11. Yeast Cell Expression

Construction of Pichia pastoris Expression Vectors for Heavy and LightChain.

The humanized variable light and heavy chain fragments were amplifiedfrom the mammalian expression vectors using PCR and subcloned into apGAP expression vector. The pGAP expression vector uses the GAP promoterto drive expression of the immunoglobulin chain and a secretion leadersequence for export. In addition, this vector contains common elementssuch as a bacterial origin of replication, and a copy of an antibioticresistance gene for selection of transformants that contain the desiredexpression vector integrated into their genome. For the vectorstargeting integration into the GAP promoter locus of the P. pastorisgenome, the pGAP vector carries an expression cassette for the kanamycinresistance gene which confers resistance to the antibiotic G418. For thevector targeting integration into the HIS4 TT locus of the P. pastorisgenome, the pGAP vector carries an expression cassette for the Sh blegene that permits selection of transformants with the antibiotic Zeocin.

Transformation of Expression Vectors into Haploid Met1 and Lys3 HostStrains of Pichia pastoris

All methods used for transformation of haploid P. pastoris strains weredone as described in Lin-Cereghino et al., Biotechniques. 2005 January;38(1):44, 46, 48. Prior to transformation each vector was linearizedwithin the GAP promoter sequences to direct the integration of thevector into the GAP promoter locus of the P. pastoris genome. Haploidstrains were transfected using electroporation and successfultransformants were selected on YPDS (yeast extract, peptone dextrosewith sorbitol) G418 agar plates. Copy numbers of heavy and light chaingenes were determined for haploid strains by Southern blot analysis.Dual locus strains were generated using the methods disclosed in U.S.Pre-Grant Patent Publication No. 2013/0045888, the contents of which areincorporated by reference in its entirety. Briefly, a haploid containingtwo copies of the heavy chain expression vector integrated at pGAP wasidentified and retransformed with a heavy chain expression vectortargeting integration into the HIS4 TT locus. Transformants containingcopies of heavy chain expression vectors integrated at both the GAPpromoter and HIS4 TT loci were selected on YPDS plates containing G418and Zeocin. Haploid strains were then mated and selected for theirability to grow in the absence of the amino acid markers (i.e., Lys andMet). Resulting diploid clones were then subjected to a final Southernblot to confirm copy numbers of heavy and light chain genes. A cloneexpressing the antibody of interest was characterized using biolayerinterferometry Protein-A biosensors to monitor expression (Octet,ForteBio).

Example 12. Expression of Ab1.H, Ab2.H, Ab3.H, Ab4.H, Ab11.H, Ab11A.H,Ab12.H, and Ab13.H in Pichia pastoris

Pichia strains for expression of full-length antibody were made. For allthe full length antibody expressing strains, haploids strains werecreated and subsequently mated. One haploid strain expressed full-lengthlight chain sequence and another haploid strain expressed thefull-length heavy chain sequence. Each diploid strain was used togenerate a research cell bank and used for expression in a bioreactor.

First an inoculum was expanded using the research cell bank using mediumcomprised of the following nutrients (% w/v): yeast extract 3%, glycerol2%, YNB 1.34%, Biotin 0.004% and 200 mM potassium phosphate. To generatethe inoculum for the fermenters, the cell bank was expanded forapproximately 29 hours in a shaking incubator at 30° C. and 300 RPM. A10% inoculum was then added to Labfors 2.5 L working volume vesselscontaining 1 L sterile growth medium. The growth medium was comprised ofthe following nutrients: potassium sulfate 18.2 g/L, ammonium phosphatemonobasic 35.6 g/L, potassium phosphate dibasic 12.8 g/L, magnesiumsulfate heptahydrate 3.72 g/L, sodium citrate dihydrate 10 g/L, glycerol40 g/L, yeast extract 30 g/L, PTM1 trace metals 4.35 mL/L, and antifoam204 1.67 mL/L. The PTM1 trace metal solution was comprised of thefollowing components: cupric sulfate pentahydrate 6 g/L, sodium iodide0.08 g/L, manganese sulfate hydrate 3 g/L, sodium molybdate dehydrate0.2 g/L, boric acid 0.02 g/L, cobalt chloride 0.5 g/L, zinc chloride 20g/L, ferrous sulfate heptahydrate 65 g/L, biotin 0.2 g/L, and sulfuricacid 5 mL/L.

The bioreactor process control parameters were set as follows: Agitation1,000 RPM, airflow 1.35 standard liter per minute, temperature 28° C.and pH was controlled at six using ammonium hydroxide. No oxygensupplementation was provided.

Fermentation cultures were grown for approximately 12 to 16 hours untilthe initial glycerol was consumed as denoted by a dissolved oxygenspike. Immediately following the dissolved oxygen spike, a bolusaddition of ethanol was added to the reactor to reach 1% ethanol (w/v).The fermentation cultures were allowed to equilibrate for 15 to 30minutes. Feed addition was initiated 30 minutes post-ethanol bolus andset at a constant rate of 0.5 mL/min for 40 minutes, then the feed pumpwas controlled by an ethanol sensor keeping the concentration of ethanolat 1% for the remainder of the run using an ethanol sensing probe (RavenBiotech). The feed was comprised of the following components: yeastextract 50 g/L, anhydrous dextrose 500 g/L, sodium citrate dehydrate 0.5g/L and PTM1 trace metals 12 mL/L. The total fermentation time wasapproximately 86 hours.

Example 13. Reduction of Corticosterone Levels in Rats by Anti-ACTHAntibodies

A pharmacodynamics study was conduced in male Lewis rats. On day 1, ratswere implanted with an Alzet pump (Durect #2ML1, 10 ul/hr for 8 days)delivering either vehicle or rat ACTH (Bachem) at a rate of 0.05mg/kg/day. Twenty-four hours later the rats were injected with either 10mg/kg of a control isotype antibody (AD26-10) or Ab1.H. Injections wereperformed by IV (tail vein) bolus administration. The study wasterminated 8 days post antibody injection.

Body weights were recorded daily and blood samples were collected on day0, 2, 3, 5, 7, and 8 in K₃EDTA tubes and processed to plasma forcorticosterone and aldosterone analysis. All samples were stored at −70°C.

Corticosterone levels in rat plasma samples were assessed using aCorticosterone EIA kit (Enzo Life Sciences) according to themanufacturer's protocol. Briefly, 100 μl plasma samples were diluted1:20, standards and controls were added to the assay plate, followed by50 μl of an alkaline phosphatase conjugated corticosterone and 50 μl ofa polyclonal Ab to corticosterone. The assay plate was incubated 2 hoursat room temperature with shaking and then washed. It was developed withp-Npp for 1 hour, then stopped and read at 405 with a 570 nmsubtraction.

Aldosterone levels in rat plasma samples were assessed using analdosterone EIA kit (Enzo Life Sciences) according to the manufacturer'sprotocol. Briefly, 100 μl plasma samples were diluted 1:10, standardsand controls were added to the assay plate, followed by 50 μl of analkaline phosphatase conjugated aldosterone and 50 μl of a polyclonal Abto aldosterone. The assay plate was incubated 16-24 hours at 4 C andthen washed. It was developed with p-Npp for 1 hour, then stopped andread at 405 with a 570 nm subtraction.

Results

FIGS. 52-64 show the effects of Ab1.H on changes in body weight, serumcorticosterone, and serum aldosterone levels that resulted from ACTHdosing. FIG. 52 shows the percentage change in animal weight by day overthe course of the study, and shows that Ab1.H inhibited ACTH-inducedweight loss. FIGS. 53 and 59 respectively show plasma corticosterone andaldosterone levels before ACTH and antibody dosing. FIGS. 54 and 60respectively show plasma corticosterone and aldosterone levels at 24hours after initiation of ACTH dosing and before the antibodyadministration. FIGS. 55 and 61 respectively show plasma corticosteroneand aldosterone levels 48 hours after initiation of ACTH dosing and 24hours after the antibody administration. FIGS. 56 and 62 respectivelyshow plasma corticosterone and aldosterone levels 96 hours afterinitiation of ACTH dosing and 72 hours after the antibodyadministration. FIGS. 57 and 63 respectively show plasma corticosteroneand aldosterone levels 144 hours after initiation of ACTH dosing and 120hours after the antibody administration. FIGS. 58 and 64 respectivelyshow plasma corticosterone and aldosterone levels 168 hours afterinitiation of ACTH dosing and 144 hours after the antibodyadministration. The results of statistical comparison between treatmentgroups at the varying time points are as indicated in the figures, andindicate statistically significant decreases in corticosterone andaldosterone caused by Ab1.H in the ACTH treatment group relative toanimals treated with the isotype control, as well as statisticallysignificant increases in corticosterone and aldosterone caused by ACTHdosing relative to vehicle-treated controls.

Overall, FIGS. 52-64 demonstrate that Ab1.H reduced corticosterone andaldosterone levels, and inhibited ACTH-induced weight loss.

Example 14. Reduction of Corticosterone Levels in Rats by Anti-ACTHAntibodies

A pharmacodynamics study was conduced in male Lewis rats. On day 1, ratswere implanted with an Alzet pump (Durect #2ML1, 10 ul/hr for 7 days)delivering either vehicle or rat ACTH (Bachem) at a rate of 0.05mg/kg/day. Twenty-fours hours later the rats were injected with either10 mg/kg of a control antibody of the same isotype (AD26-10), Ab7A.HAb10.H, Ab11.H, Ab12.H, Ab13.H, Ab15.H, Ab11A.H, or Ab15.H, or withAb2.H at 100 mg/kg Injections were performed by IV (tail vein) bolusadministration. The study was terminated 7 days post antibody injection.

Body weights were recorded daily and blood samples were collected on day0, 2, 3, 5, and 7 in K₃EDTA tubes and processed to plasma forcorticosterone and aldosterone analysis. All samples were stored at −70°C.

Corticosterone levels in rat plasma samples were assessed using aCorticosterone EIA kit (Enzo Life Sciences) according to manufacturer'sprotocol. Briefly 100 μl plasma samples were diluted 1:100, standardsand controls were added to assay plate, followed by 50 μl of an alkalinephosphatase conjugated corticosterone and 50 μl of a polyclonal Ab tocorticosterone. The assay plate was incubated 2 hours at roomtemperature with shaking and then washed. It was developed with p-Nppfor 1 hour, then stopped and read at 405 with a 570 nm subtraction.

Aldosterone levels in rat plasma samples were assessed using analdosterone EIA kit (Enzo Life Sciences) according to the manufacturer'sprotocol. Briefly, 100 μl plasma samples were diluted 1:10, standardsand controls were added to the assay plate, followed by 50 μl of analkaline phosphatase conjugated aldosterone and 50 μl of a polyclonal Abto aldosterone. The assay plate was incubated 16-24 hours at 4 C andthen washed. It was developed with p-Npp for 1 hour, then stopped andread at 405 with a 570 nm subtraction.

Results

FIGS. 65-75 show the effects of Ab2.H, Ab11.H, and Ab12.H on changes inbody weight, serum corticosterone, and serum aldosterone levels thatresulted from ACTH dosing. FIG. 65 shows the percentage change in animalweight by day over the course of the study, and shows that Ab2.H,Ab11.H, and Ab12.H inhibited ACTH-induced weight loss. FIGS. 66 and 71respectively show plasma corticosterone and aldosterone levels beforeACTH and antibody dosing. FIGS. 67 and 72 respectively show plasmacorticosterone and aldosterone levels at 24 hours after initiation ofACTH dosing and before the antibody administration. FIGS. 68 and 73respectively show plasma corticosterone and aldosterone levels 48 hoursafter initiation of ACTH dosing and 24 hours after the antibodyadministration. FIGS. 69 and 74 respectively show plasma corticosteroneand aldosterone levels 96 hours after initiation of ACTH dosing and 72hours after the antibody administration. FIGS. 70 and 75 respectivelyshow plasma corticosterone and aldosterone levels 144 hours afterinitiation of ACTH dosing and 120 hours after the antibodyadministration. The results of statistical comparison between treatmentgroups at the varying time points are as indicated in the figures, andindicate statistically significant decreases in corticosterone andaldosterone caused by Ab2.H, Ab11.H, and Ab12.H in the ACTH treatmentgroup relative to animals treated with the isotype control, as well asstatistically significant increases in aldosterone caused by ACTH dosingrelative to vehicle-treated controls.

Overall, FIGS. 65-75 demonstrate that Ab2.H, Ab1.H, and Ab12.H inhibitedACTH-induced weight loss and ACTH-induced increases in corticosteroneand aldosterone levels.

FIGS. 76-86 show the effects of Ab10.H on changes in body weight, serumcorticosterone, and serum aldosterone levels that resulted from ACTHdosing. FIG. 76 shows the percentage change in animal weight by day overthe course of the study, and shows that Ab10.H inhibited ACTH-inducedweight loss. FIGS. 77 and 82 respectively show plasma corticosterone andaldosterone levels before ACTH and antibody dosing. FIGS. 78 and 83respectively show plasma corticosterone and aldosterone levels at 24hours after initiation of ACTH dosing and before the antibodyadministration. FIGS. 79 and 84 respectively show plasma corticosteroneand aldosterone levels 48 hours after initiation of ACTH dosing and 24hours after the antibody administration. FIGS. 80 and 85 respectivelyshow plasma corticosterone and aldosterone levels 96 hours afterinitiation of ACTH dosing and 72 hours after the antibodyadministration. FIGS. 81 and 86 respectively show plasma corticosteroneand aldosterone levels 144 hours after initiation of ACTH dosing and 120hours after the antibody administration. The results of statisticalcomparison between treatment groups at the varying time points are asindicated in the figures, and indicate statistically significantdecreases in corticosterone and aldosterone caused by Ab10.H in the ACTHtreatment group relative to animals treated with the isotype control, aswell as statistically significant increases in corticosterone andaldosterone caused by ACTH dosing relative to vehicle-treated controls.

Overall, FIGS. 76-86 demonstrate that Ab10.H inhibited ACTH-inducedweight loss and ACTH-induced increases in corticosterone and aldosteronelevels.

FIGS. 87-97 show the effects of Ab13.H on changes in body weight, serumcorticosterone, and serum aldosterone levels that resulted from ACTHdosing. FIG. 87 shows the percentage change in animal weight by day overthe course of the study, and shows that Ab13.H inhibited ACTH-inducedweight loss. FIGS. 88 and 93 respectively show plasma corticosterone andaldosterone levels before ACTH and antibody dosing. FIGS. 89 and 94respectively show plasma corticosterone and aldosterone levels at 24hours after initiation of ACTH dosing and before the antibodyadministration. FIGS. 90 and 95 respectively show plasma corticosteroneand aldosterone levels 48 hours after initiation of ACTH dosing and 24hours after the antibody administration. FIGS. 91 and 96 respectivelyshow plasma corticosterone and aldosterone levels 96 hours afterinitiation of ACTH dosing and 72 hours after the antibodyadministration. FIGS. 92 and 97 respectively show plasma corticosteroneand aldosterone levels 144 hours after initiation of ACTH dosing and 120hours after the antibody administration. The results of statisticalcomparison between treatment groups at the varying time points are asindicated in the figures, and indicate statistically significantdecreases in corticosterone and aldosterone caused by Ab13.H in the ACTHtreatment group relative to animals treated with the isotype control, aswell as statistically significant increases in corticosterone andaldosterone caused by ACTH dosing relative to vehicle-treated controls.

Overall, FIGS. 87-97 demonstrate that Ab13.H inhibited ACTH-inducedweight loss and ACTH-induced increases in corticosterone and aldosteronelevels.

FIGS. 98-108 show the effects of Ab7A.H (labeled in the figure asAb7A.H) on changes in body weight, serum corticosterone, and serumaldosterone levels that resulted from ACTH dosing. FIG. 98 shows thepercentage change in animal weight by day over the course of the study,and shows that Ab7A.H inhibited ACTH-induced weight loss. FIGS. 99 and104 respectively show plasma corticosterone and aldosterone levelsbefore ACTH and antibody dosing. FIGS. 100 and 105 respectively showplasma corticosterone and aldosterone levels at 24 hours afterinitiation of ACTH dosing and before the antibody administration. FIGS.101 and 106 respectively show plasma corticosterone and aldosteronelevels 48 hours after initiation of ACTH dosing and 24 hours after theantibody administration. FIGS. 102 and 107 respectively show plasmacorticosterone and aldosterone levels 96 hours after initiation of ACTHdosing and 72 hours after the antibody administration. FIGS. 103 and 108respectively show plasma corticosterone and aldosterone levels 144 hoursafter initiation of ACTH dosing and 120 hours after the antibodyadministration. The results of statistical comparison between treatmentgroups at the varying time points are as indicated in the figures, andindicate statistically significant decreases in corticosterone caused byAb7A.H in the ACTH treatment group relative to animals treated with theisotype control, as well as statistically significant increases incorticosterone caused by ACTH dosing relative to vehicle-treatedcontrols.

Overall, FIGS. 98-108 demonstrate that Ab7A.H inhibited ACTH-inducedweight loss and ACTH-induced increases in corticosterone and aldosteronelevels.

FIGS. 109-112 show the effects of Ab11A.H and Ab15.H on changes in serumcorticosterone levels that resulted from ACTH dosing. FIG. 109 showsplasma corticosterone levels before ACTH and antibody dosing. FIG. 110shows plasma corticosterone levels at 24 hours after initiation of ACTHdosing and before the antibody administration. FIG. 111 shows plasmacorticosterone levels 48 hours after initiation of ACTH dosing and 24hours after the antibody administration. FIG. 112 shows plasmacorticosterone levels 96 hours after initiation of ACTH dosing and 72hours after the antibody administration. The results of statisticalcomparison between treatment groups at the varying time points are asindicated in the figures, and indicate statistically significantdecreases in corticosterone caused by Ab11A.H and Ab15.H in the ACTHtreatment group relative to animals treated with the isotype control, aswell as statistically significant increases in corticosterone caused byACTH dosing relative to vehicle-treated controls.

Overall, FIGS. 109-112 demonstrate that Ab11A.H and Ab15.H inhibitedACTH-induced increases in corticosterone levels.

Example 15. Pharmacokinetic Study of Ab13.H in Rats

A pharmacokinetic (PK) study was conducted in male Sprague-Dawley ratssurgically implanted with a jugular vein catheter. Four rats were dosedwith Ab13.H (5 mg/kg) by IV bolus injection on day 1. Blood samples werecollected (into K₃EDTA tubes) via the jugular vein catheter prior toantibody dosing, 0.25, 0.5, 1, 2, 8, 12, 24, 48, 96, 144, 192, 312, 384,and 480 hours post dose administration. Whole blood samples wereprocessed to plasma for total antibody level determination. Theconcentration of total antibody (Ab13.H) in rat plasma was determinedusing a direct ELISA sandwich format. Briefly, diluted standards,controls, and study samples in 10% rat plasma matrix were incubated withgoat anti-Human IgG, which had been immobilized on MSD High Bindmicrotiter plates. After incubation, unbound material was washed awayand Ab13.H was detected with biotinylated goat anti-Human IgG (H+L) andSulfo-TAG Streptavidin. After washing, diluted 1× Read Buffer was addedto each well. Electrochemiluminescence (ECL) signals were measured byMSD SECTOR Imager 2400. Subsequently, ECL signals were collected andanalyzed using the MSD Discovery Workbench software. A standard curvewas generated using a 4-parameter fit algorithm for each plate. Theconcentration of total Ab13.H was determined by comparing the ECL signalto the corresponding standard curve. Plasma drug level data underwentpharmacokinetic evaluation using WinNonLin (Professional version 4.1;Pharsight Corporation, Mountain View, Calif.).

Results

FIG. 113 shows total Ab13.H antibody levels determined in each rat.WinNonLin analysis of total antibody levels and computed pharmacokineticparameters is presented in Table 11.

TABLE 11 WinNonLin analysis of pharmacokinetic parameters for Ab13.Hplasma levels in the rat. Animal HL_Lambda_z Tmax Cmax AUCINF_predVz_pred Cl_pred Vss_pred No. (hr) (hr) (ug/mL) (hr*ug/mL) (mL/kg)(mL/hr/kg) (mL/kg) 1 159.21 0.25 72.99 4654.00 246.77 1.07 221.91 2169.00 0.25 68.75 4469.27 272.77 1.12 243.62 3 102.82 0.50 58.42 3396.59218.37 1.47 194.07 4 103.15 0.25 53.62 3251.91 228.81 1.54 212.36 N 4 44 4 4 4 4 Mean 133.546 0.313 63.445 3942.942 241.679 1.301 217.992 SD35.513 0.125 8.963 720.799 23.814 0.238 20.623

Example 16. Reduction of Corticosterone Levels in Cynomolgus Monkeys byAb13.H

A pharmacodynamics (PD) study was conducted in male cynomolgus monkeys.Three cynomolgus monkeys were injected with either Ab13.H or an isotypecontrol antibody (AD26-10) at 10 mg/kg by IV bolus administration on day1 and day 7. At each time point three blood samples were collected byvenipuncture: one sample was processed to serum for clinical chemistryanalysis; one sample processed to plasma for cortisol and aldosteronedetermination; and whole blood was used for CBC analyses. Blood sampleswere collected day −6 (6 days prior to the first antibody dose), day 3,day 7 (prior to second antibody dose), day 9, day 13, day 17 and day 21.Clinical chemistry analyses included the following parameters: alanineaminotransferase, aspartate aminotransferase, alkaline phosphatase,gamma-glutamyltransferase, lactate dehydrogenase, total bilirubin, ureanitrogen, creatinine, calcium, phosphorus, total protein, albumin,globulin, albumin/globulin ratio, glucose, cholesterol, triglycerides,sodium, potassium, and chloride.

Results: No significant changes were seen in clinical chemistryparameters

Blood draws were taken (into K₃EDTA collection tubes) at the same timespoints as described above from clinical chemistry analyses and thefollowing hematology parameters determined: red blood cell count,hemoglobin concentration, hematocrit, mean corpuscular volume, red bloodcell distribution width, mean corpuscular hemoglobin concentration,reticulocyte count, platelet count, white blood cell count, neutrophilcount, lymphocyte count, monocyte count, eosinophil count, basophilcount, and large unstained cells.

Results: No significant changes were seen in hematology parameters.

Blood draws were taken (K₃EDTA) and processed to plasma for cortisol andaldosterone levels determined. Cortisol levels in cynomolgus monkeyplasma samples were assessed using a Cortisol EIA kit (R&D) essentiallyaccording to the manufacturer's protocol. Briefly 50 ul of a primaryantibody was incubated on the plate for 1 hour with shaking, the platewas washed as directed and then 100 μl of plasma samples diluted 1:100,standards, and controls were added to the assay plate, followed by 50 μlof an HRP conjugated cortisol. The assay plate was incubated 2 hours atroom temperature with shaking and then washed. It was developed with TMBfor 1 hour, then stopped and read at 450 with a 540 nm subtraction.

Results: FIG. 114 demonstrates that Ab13.H reduced cortisol levels incynomolgus monkeys.

Aldosterone levels in cynomolgus monkey plasma samples were assessedusing an aldosterone EIA kit (Enzo Life Sciences) according to themanufacturer's protocol. Briefly 100 μl plasma samples diluted 1:10,standards, and controls were added to the assay plate, followed by 50 μlof an alkaline phosphatase conjugated aldosterone and 50 μl of apolyclonal antibody to aldosterone. The assay plate was incubated 16-24hours at 4 C and then washed. It was developed with p-Npp for 1 hour,then stopped and read at 405 with a 570 nm subtraction.

Results: FIG. 115 demonstrates that Ab13.H modestly reduced andmodulated aldosterone levels in cynomolgus monkeys.

Example 17. Reduction of Corticosterone Levels in Rats by Ab13.H

A pharmacodynamic study was conducted in male Lewis rats. Groups of 8rats each were injected with either a control isotype antibody (AD26-10)at 10 mg/kg or Ab13.H at 10 mg/kg, 1 mg/kg, 0.1 mg/kg or 0.01 mg/kg. Allinjections were performed by IV bolus administration on day 0. Two bloodsamples were collected before test article administration (day −3 andday 0) and 6 blood samples collected post test article administration(day 1, 3, 6, 9, 12 and 15). All blood samples were collected in K₃EDTAtubes and processed into plasma for corticosterone analysis.

Corticosterone levels in rat plasma samples were assessed using aCorticosterone EIA kit (Enzo Life Sciences) according to manufacturer'sprotocol. Briefly, 100 μl plasma samples diluted 1:50, standards andcontrols were added to assay plate, followed by 50 μl of an alkalinephosphatase conjugated corticosterone and 50 μl of a polyclonal Ab tocorticosterone. Assay plates were incubated 2 hours at room temperaturewith shaking and then washed. Plates were developed with pNpp substratefor 1 hour and read at 405 with a 570 nm subtraction.

The concentration of total antibody (AD26-10 and Ab13.H) in rat plasmawas determined using a direct ELISA sandwich format. Briefly, dilutedstandards, controls, and study samples in 10% rat plasma matrix wereincubated with goat anti-Human IgG, which had been immobilized on MSDHigh Bind microtiter plates. After incubation, unbound material waswashed away and Ab13.H was detected with biotinylated goat anti-HumanIgG (H+L) and Sulfo-TAG Streptavidin. After washing, diluted 1× ReadBuffer was added to each well. Electrochemiluminescence (ECL) signalswere measured by MSD SECTOR Imager 2400. Subsequently, ECL signals werecollected and analyzed using the MSD Discovery Workbench software. Astandard curve was generated using a 4-parameter fit algorithm for eachplate. The concentration of total antibody was determined by comparingthe ECL signal to the corresponding standard curve.

Results: FIG. 116 shows the corticosterone levels observed in rats dosedwith AD26-10 or Ab13.H. Injection of Ab13.H at 10 mg/kg or 1 mg/kgsignificantly decreased corticosterone levels at all time points postadministration (p<0.05). Injection of Ab13.H at 0.1 mg/kg significantlydecreased corticosterone levels on days 1 and 3 days post administration(p<0.05) and then levels were similar to control animals. Administrationof Ab13.H at 0.01 mg/kg did not significantly alter corticosteronelevels.

FIG. 117 shows the total antibody levels following administration ofAD26-10 or Ab13.H. Total antibody levels of AD26-10 and Ab13.H in ratsdosed with 10 mg/kg are comparable and Ab13.H dosed at 1 mg/kg, 0.1mg/kg and 0.01 mg/kg are present at levels consistent with the dose.

Example 18. Reduction of Corticosterone Levels in Rabbits by Ab13.H

A pharmacodynamics study was conducted in male New Zealand Whiterabbits. On day 1, two groups of five rabbits each were injected witheither Ab13.H (50 mg/kg) or vehicle by IV bolus administration. On day10, the environment of the rabbits was changed from open to the naturalenvironment to a room with an ambient temperature of 20° C. and a 12hour light/darkness cycle. The rabbits remained in the alteredenvironmental conditions for 31.5 hours and then were returned to theoriginal housing conditions. The change in environment was performed toplace stress on the animals which was expected to induce corticosteronevia an ACTH-dependent pathway. On day 15, the rabbits were injected IVwith 15 μg rabbit ACTH (American Peptide). The study was terminated onday 15.

Blood samples were collected on days −7, −3, 2, 3, 7, 10, 11, and 15 (1hour post ACTH injection) in K₃ETDA tubes and processed to plasma forcorticosterone analysis. All samples were stored at −70° C. prior toanalysis.

Corticosterone levels in rabbit plasma samples were measured using aCorticosterone EIA kit (Enzo Life Sciences) following the manufacturer'sinstructions. Briefly, 100 μl plasma samples were diluted 1:10,standards and controls were added to assay plate, followed by 50 μl ofan alkaline phosphatase conjugated corticosterone and 50 μl of apolyclonal antibody to corticosterone. The assay plate was incubated 2hours at room temperature with shaking and then washed. It was developedwith p-Npp for 1 hour, then stopped and read at 405 with a 570 nmsubtraction.

Results: FIG. 118 shows the plasma corticosterone levels in the twogroups over the study period. The Ab13.H treatment group exhibitedreduced corticosterone levels on days 2, 3 and 7 compared to controlrabbits. On days 10 and 11, rabbits were subjected to alteredenvironmental conditions that modestly induced corticosterone levels inthe control rabbits, and corticosterone levels in Ab13.H treatment groupwere slightly induced but to a lesser degree than control animals. Onday 15, rabbits were injected with 15 μg rabbit ACTH, which dramaticallyinduced corticosterone in control animals. Corticosterone levels on day15 in the rabbits in the Ab13.H treatment group were only modestlyinduced and were significantly lower than control rabbits (p=0.0079).

Example 19. Reduction of Corticosterone Levels by Ab13.H in Rats UnderLow and High Stress

A pharmacodynamics study was conducted in male Lewis rats subjected toblood draws under low and high stress conditions. Low stress blood drawswere performed by placing the rats in anesthesia chambers that werelightly heated using heat pads for approximately 5 minutes. Isofluranewas introduced into the chamber for 7-10 minutes and rats were bledafter losing consciousness. High stress blood draws were performed byplacing the rats in a plexiglass cylinder rat restraint with the ratsheld at maximum pressure and lightly heated using heat pads forapproximately 10 minutes. Rats were then bled without anesthesia via thetail vein using the same rat restrainer. Ab13.H or a control isotypeantibody (AD26-10) was administered at 10 mg/kg IV on day 0.

Blood draws were collected on days −5 and −2 prior to antibodyadministration using the low stress procedure. Blood draws werecollected on days 2 and 6 using the low stress procedure and days 3 and5 using the high stress procedure. Blood draws were collected in K₃EDTAtubes and processed to plasma for corticosterone analysis. All sampleswere stored at −70° C. until analysis.

Corticosterone levels in rat plasma samples were assessed using aCorticosterone EIA kit (Enzo Life Sciences) following the manufacturer'sinstructions. Briefly, 100 μl plasma samples were diluted 1:20,standards and controls were added to assay plate, followed by 50 μl ofan alkaline phosphatase conjugated corticosterone and 50 μl of apolyclonal antibody to corticosterone. The assay plate was incubated 2hours at room temperature with shaking and then washed. It was developedwith p-Npp for 1 hour, then stopped and read at 405 with a 570 nmsubtraction.

Results: FIG. 119 shows the plasma corticosterone levels in rats dosedwith AD26-10 (square symbols) or Ab13.H (round symbols) and subjected tolow or high stress conditions. Administration of Ab13.H reducedcorticosterone levels to below the lower limit of quantitation (LLOQ)under the low stress blood draw conditions (days 2 and 6). The highstress blood draw conditions induced corticosterone in the controlanimals. The induction of corticosterone under the high stress blooddraw conditions in the Ab13.H treated rats was measurable but wassignificantly lower than control rats. In the Ab13.H treatment group,corticosterone levels were statistically significantly reduced on days2, 3, 5 (p=0.0002) and 6 (p=0.0068) compared to the control group.

The above description of various illustrated embodiments of theinvention is not intended to be exhaustive or to limit the invention tothe precise form disclosed. While specific embodiments of, and examplesfor, the invention are described herein for illustrative purposes,various equivalent modifications are possible within the scope of theinvention, as those skilled in the relevant art will recognize. Theteachings provided herein of the invention can be applied to otherpurposes, other than the examples described above.

These and other changes can be made to the invention in light of theabove detailed description. In general, in the following claims, theterms used should not be construed to limit the invention to thespecific embodiments disclosed in the specification and the claims.Accordingly, the invention is not limited by the disclosure, but insteadthe scope of the invention is to be determined entirely by the followingclaims.

The invention may be practiced in ways other than those particularlydescribed in the foregoing description and examples. Numerousmodifications and variations of the invention are possible in light ofthe above teachings and, therefore, are within the scope of the appendedclaims.

What is claimed is:
 1. A nucleic acid construct comprising a nucleicacid encoding a variable heavy chain (VH) or heavy chain polypeptide ofan anti-human adrenocorticotrophic hormone (ACTH) antibody, wherein theencoded VH or heavy chain polypeptide comprises heavy chain CDR1, CDR2,and CDR3 polypeptides comprising the amino acid sequences of SEQ ID NO:564; SEQ ID NO: 566; and SEQ ID NO: 568, respectively, and comprises atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:562.
 2. The nucleic acid construct according to claim 1, wherein theencoded VH polypeptide comprises the amino acid sequence of SEQ ID NO:562.
 3. The nucleic acid construct according to claim 1, wherein theencoded heavy chain polypeptide comprises the amino acid sequence of SEQID NO:
 561. 4. The nucleic acid construct according to claim 1, whichcomprises the nucleic acid sequence of SEQ ID NO: 572 or
 571. 5. Avector comprising the nucleic acid construct according to claim
 1. 6. Anucleic acid construct comprising a nucleic acid encoding a variablelight chain (VL) or light chain polypeptide of an anti-human ACTHantibody, wherein the encoded VL or light chain polypeptide compriseslight chain CDR1, CDR2, and CDR3 polypeptides comprising the amino acidsequences of SEQ ID NO: 584; SEQ ID NO: 586; and SEQ ID NO: 588,respectively, and comprises at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:
 582. 7. The nucleic acid construct accordingto claim 6, wherein the encoded VL polypeptide comprises the amino acidsequence of SEQ ID NO:
 582. 8. The nucleic acid construct according toclaim 6, wherein the encoded light chain polypeptide comprises the aminoacid sequence of SEQ ID NO:
 581. 9. The nucleic acid construct accordingto claim 6, which comprises the nucleic acid sequence of SEQ ID NO: 592or
 591. 10. A vector comprising a nucleic acid construct according toclaim
 6. 11. A nucleic acid construct comprising: (a) a nucleic acidencoding a VII or heavy chain polypeptide of an anti-human ACTHantibody, wherein the encoded VH or heavy chain polypeptide comprisesheavy chain CDR1, CDR2, and CDR3 polypeptides comprising the amino acidsequences of SEQ ID NO: 564; SEQ ID NO: 566; and SEQ ID NO: 568,respectively, and comprises at least 90% sequence identity to the aminoacid sequence of SEQ ID NO: 562; and (b) a nucleic acid encoding a VL orlight chain polypeptide of an anti-human ACTH antibody, wherein theencoded VL or light chain polypeptide comprises light chain CDR1, CDR2,and CDR3 polypeptides comprising the amino acid sequences of SEQ ID NO:584; SEQ ID NO: 586; and SEQ ID NO: 588, respectively, and comprises atleast 90% sequence identity to the amino acid sequence of SEQ ID NO:582.
 12. The nucleic acid construct according to 11, wherein: (a) theencoded VH polypeptide comprises the amino acid sequence of SEQ ID NO:562; and (b) the encoded VL polypeptide comprises the amino acidsequence of SEQ ID NO:
 582. 13. The nucleic acid construct according to11, wherein: (a) the encoded heavy chain polypeptide comprises the aminoacid sequence of SEQ ID NO: 561; and (b) the encoded light chainpolypeptide comprises the amino acid sequence of SEQ ID NO:
 581. 14. Avector comprising the nucleic acid construct according to claim
 13. 15.The nucleic acid construct according to 11, wherein: (a) the nucleicacid encoding the VH or heavy chain polypeptide comprises the nucleicacid sequence of SEQ ID NO: 572 or 571, respectively: and (b) thenucleic acid encoding the VL or light chain polypeptide comprises thenucleic acid sequence of SEQ ID NO: 592 or 591, respectively.
 16. Avector comprising the nucleic acid construct according to claim
 11. 17.The nucleic acid construct according to claim 11, which encodes a scFvcomprising said VH and VL polypeptides.
 18. The nucleic acid constructaccording to claim 11, wherein the encoded constant domains are theconstant domains of a human IgG1, IgG2, IgG3, or IgG4.
 19. The nucleicacid construct according to claim 11, wherein the encoded VH and VLpolypeptides are humanized.
 20. A host cell comprising: (a) a nucleicacid which encodes a heavy chain polypeptide which comprises heavy chainCDR1, CDR2, and CDR3 polypeptides comprising the amino acid sequences ofSEQ ID NO: 564; SEQ ID NO: 566; and SEQ ID NO: 568, respectively; and(b) a nucleic acid which encodes a light chain polypeptide whichcomprises light chain CDR1, CDR2, and CDR3 polypeptides comprising theamino acid sequences of SEQ ID NO: 584; SEQ ID NO: 586; and SEQ ID NO:588, respectively, wherein both nucleic acid (i) and nucleic acid (ii)are operably linked to a promoter and further wherein the expression ofnucleic acid (i) and nucleic acid (ii) in said host cell results in ananti-human ACTH antibody.
 21. The host cell according to claim 20, whichcomprises a bacterial, yeast, insect or mammalian cell.
 22. The hostcell according to claim 20, which comprises a CHO or Pichia pastoriscell.
 23. A method of producing an anti-human ACTH antibody, wherein themethod comprises culturing the host cell according to claim 20 underconditions that result in the expression of said anti-human ACTHantibody.
 24. The method of claim 23, wherein the expressed ACTHantibody comprises: (a) a VH polypeptide which comprises the amino acidsequence of SEQ ID NO: 562; and (b) a VL polypeptide which comprises theamino acid sequence of SEQ ID NO:
 582. 25. The method of claim 23,wherein the expressed ACTH antibody comprises: (a) a heavy chainpolypeptide which comprises the amino acid sequence of SEQ ID NO: 561;and (b) a light chain polypeptide which comprises the amino acidsequence of SEQ ID NO:
 581. 26. A method of producing an anti-human ACTHFab fragment, wherein the method comprises: (i) culturing the host cellaccording to claim 20 under conditions that result in the expression ofsaid anti-human ACTH antibody; and (ii) enzymatically digesting theexpressed anti-human ACTH antibody, thereby producing the anti-humanACTH Fab fragment.